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REVIEW ARTICLE OPEN

Targeting cancer stem cell pathways for cancer therapy

Liqun Yang1,2, Pengfei Shi1,2, Gaichao Zhao1,2, Jie Xu1,2, Wen Peng1,2, Jiayi Zhang1,2, Guanghui Zhang1,2, Xiaowen Wang1,2,
Zhen Dong1,2, Fei Chen3 and Hongjuan Cui1,2

Since cancer stem cells (CSCs) were first identified in leukemia in 1994, they have been considered promising therapeutic targets for
cancer therapy. These cells have self-renewal capacity and differentiation potential and contribute to multiple tumor malignancies,
such as recurrence, metastasis, heterogeneity, multidrug resistance, and radiation resistance. The biological activities of CSCs are
regulated by several pluripotent transcription factors, such as OCT4, Sox2, Nanog, KLF4, and MYC. In addition, many intracellular
signaling pathways, such as Wnt, NF-κB (nuclear factor-κB), Notch, Hedgehog, JAK-STAT (Janus kinase/signal transducers and
activators of transcription), PI3K/AKT/mTOR (phosphoinositide 3-kinase/AKT/mammalian target of rapamycin), TGF (transforming
growth factor)/SMAD, and PPAR (peroxisome proliferator-activated receptor), as well as extracellular factors, such as vascular niches,
hypoxia, tumor-associated macrophages, cancer-associated fibroblasts, cancer-associated mesenchymal stem cells, extracellular
matrix, and exosomes, have been shown to be very important regulators of CSCs. Molecules, vaccines, antibodies, and CAR-T
(chimeric antigen receptor T cell) cells have been developed to specifically target CSCs, and some of these factors are already
undergoing clinical trials. This review summarizes the characterization and identification of CSCs, depicts major factors and
pathways that regulate CSC development, and discusses potential targeted therapy for CSCs.
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Signal Transduction and Targeted Therapy (2020)5:8 ; https://doi.org/10.1038/s41392-020-0110-5

INTRODUCTION
Cancers are chronologic diseases that seriously threaten human
life. Many strategies have been developed for cancer treatment,
including surgery, radiotherapy, chemotherapy, and targeted
therapy. Because of all these treatments, the incidence rate of
cancer has been stable in women and has declined slightly in
men in the past decade (2006–2015), and the cancer death rate
(2007–2016) also declined.1 However, traditional cancer treat-
ment methods are effective only for some malignant tumors.2
The main reasons for the failure of cancer treatment are
metastasis, recurrence, heterogeneity, resistance to chemother-
apy and radiotherapy, and avoidance of immunological surveil-
lance.3 All these failures could be explained by the characteristics
of cancer stem cells (CSCs).4 CSCs can cause cancer relapse,
metastasis, multidrug resistance, and radiation resistance through
their ability to arrest in the G0 phase, giving rise to new tumors.5
Therefore, CSCs could be considered the most promising targets
for cancer treatment.
CSCs were first identified in leukemia and then isolated via
CD34+ and CD38− surface marker expression in the 1990s.6,7 CSCs
expressing different surface markers, such as CD133, nestin, and
CD44, have been subsequently found in many nonsolid and solid
tumors, and these cells also form the bulk of the tumor.8,9 CSCs
can generate tumors via the self-renewal and differentiation into
multiple cellular subtypes.10 The activities of CSCs are controlled
by many intracellular and extracellular factors, and these factors
can be used as drug targets for cancer treatment.11 To understand
the nature of CSCs, we summarized their characteristics, methods
for identification and isolation, regulation and current research on
targeting CSCs for cancer therapy both in basic research and
clinical studies.
THE CONCEPT OF CSCS
Biological characteristics of CSCs
With the deepening of tumor biology research, clinical diagnosis
and cancer treatment have significantly improved in recent years.
However, the high recurrence rate and high mortality rate are still
unresolved and are closely related to the biological characteristics
of CSCs. With further understanding of CSC characteristics,
research on tumor biology has entered a new era. Therefore,
understanding the biological properties of CSCs is of great
significance in the diagnosis and treatment of tumors.
CSCs have a strong self-renewal ability, which is the direct cause
of tumorigenesis.12 CSCs can symmetrically divide into two CSCs
or into one CSC and one daughter cell.13 CSCs expand in a
symmetrical splitting manner to excessively increase cell growth,
ultimately leading to tumor formation.14 CSCs isolated from
original tumor tissue that were transplanted into severe combined
immunodeficiency disease (SCID) mice then formed new tumors.15
CSCs and normal stem cells also share some of the same
regulatory signaling pathways, such as the Wnt/β-catenin,16 Sonic
Hedgehog (Hh),17 and Notch pathways, which are involved in the
self-renewal process.18 In addition, other signaling molecules, such
as PTEN and the polycomb family, also play important roles in the
regulation of CSC growth.19 The regulation of CSC self-renewal is
the key link to understanding tumorigenesis. These studies will
provide a clear target for cancer treatment.

1
State Key Laboratory of Silkworm Genome Biology, Southwest University, 400716 Chongqing, China; 2Cancer Center, Medical Research Institute, Southwest University, 400716
Chongqing, China and 3Department of Pharmaceutical Sciences, Eugene Applebaum College of Pharmacy and Health Sciences, Wayne State University, Detroit, MI 48201, USA
Correspondence: Hongjuan Cui (hongjuan.cui@gmail.com)
These authors contributed equally: Liqun Yang, Pengfei Shi

Received: 5 October 2019 Revised: 15 December 2019 Accepted: 19 December 2019

© The Author(s) 2020

 Targeting cancer stem cell pathways for cancer therapy
Yang et al.
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In addition to their self-renewal ability, CSCs also have the
ability to differentiate into different cell types. Bonnet and Dick7
demonstrated in 1997 that CD34+/CD38− leukemia stem cells
(LSCs) have the ability to differentiate and proliferate in SCID mice.
Brain CSCs isolated from patients are positive for the markers
CD133 and nestin, which are the same markers as those of normal
neuronal stem cells, but some cells lack surface markers for
differentiation.20 Generally, various signaling pathways regulate
the self-renewal and differentiation of normal stem cells to
promote their proliferation and differentiation in a relatively
balanced manner. Once the regulatory balance is destroyed,
uncontrolled CSCs ultimately lead to tumorigenesis.21 CSCs also
transdifferentiate into other multilineage cells to regulate
tumorigenesis.22 Bussolati et al.23 found that renal CSCs differ-
entiated into vascular endothelial cells (ECs) in the bulk of tumors
formed in SCID mice after injection of human renal CSCs.
Additionally, CSCs that differentiate into vascular ECs and promote
angiogenesis have been found in a variety of cancers, such as
glioblastoma24 and liver cancer.25
Metastasis refers to the process by which cancer cells travel
from the primary site through lymphatic vessels, blood vessels, or
the body cavity.26 Since stromal cells (such as granulocytes and
macrophages) secrete signaling molecules in the tumor micro-
environment (TME), these cells stimulate epithelial–mesenchymal
transformation (EMT) to promote the invasion of tumor cells,27
which induce differentiated human mammary epithelial cells to
form mammary glands.28 Activation of the RAS/MAPK (mitogen-
activated protein kinase) signaling pathway transforms nontu-
morigenic CD44−/CD24+ breast cancer cells into tumorigenic
CD44+/CD24− breast cancer cells.29 A study showed that CSCs are
closely related to EMT, and EMT is likely to be the basis for tumor
invasion and metastasis. In addition, CD133+/CXCR4+ pancreatic
cancer cells30 and CD44+/α2βhi1/CD133+ prostate cancer cells31
are also tumorigenic. Therefore, these studies indicate that CSCs
play a crucial role in tumor metastasis and development.
Furthermore, understanding the mechanism of CSC drug
resistance is vital for cancer treatment and preventing recur-
rence.32 CSCs efficiently express ATP-binding cassette (ABC)
transporters (including MDR1 (ABCB1), MRP1 (ABCC1), and
(ABCG2)), which are multidrug resistance proteins, and these
proteins protect leukemia and some solid tumor cells from drug
damage and induce drug resistance.33 According to previous
studies, aldehyde dehydrogenase (ALDH), a marker in many
CSCs,34 eliminates oxidative stress and enhances resistance to
chemotherapeutic drugs, such as oxazolidine, taxanes, and
platinum drugs.35 ALDH also removes free radicals induced by
radiation and stimulates resistance to radiation.35 Inducing DNA
damage and apoptosis through chemotherapy and radiotherapy
are commonly used cancer treatments. However, CSCs can
effectively protect cancer cells from apoptosis by activating DNA
repair abilities.36
It is currently believed that CSCs are the key "seeds" for tumor
initiation and development, metastasis, and recurrence.37 CSCs
have evolved and are highly heterogeneous.38 Breast CSCs have
different expression patterns of surface biomarkers, such as
CD44+, CD24−, SP, and ALDH+.29,34,39 CD271− or CD271+
melanoma stem cells can form tumors in SCID mice.40 The
heterogeneity of CSCs has also been found in other cancers,
including glioblastoma,41 prostate cancer,42 and lung cancer.43
The heterogeneity of CSCs is so complex that more effective
biomarkers are needed to identify CSCs or distinguish the
heterogeneity of CSCs.
Isolation and identification of CSCs
It is known that the proportion of CSCs in tumor tissues is very low
and generally accounts for only 0.01–2% of the total tumor mass.
In addition, CSCs and normal stem cells also share similar
transcription factors and signaling pathways. Therefore, it is more
challenging to isolate and identify CSCs. However, an increasing
number of techniques and means have emerged.
CSCs have been identified through different biomarkers in
human cancers (Table 1). CSCs can be separated by combining
specific biomarkers that are mostly located on the cell surface.3
The primary separation techniques are fluorescence-activated cell
sorting (FACS) and magnetic-activated cell sorting (MACS).44,45
Since Dick JE first screened CSCs from leukemia by using FACS
technology,7 FACS has become the most widely used technique
for cell separation. It can perform multibiomarker sorting at one
time and has high purity and strong specificity. MACS is a MACS
technique. MACS separation is relatively simple, but the technique
is cumbersome. Therefore, this method requires high activity of
CSCs.44,46 These two methods are effective in separating CSCs
from large numbers of cells.
Additionally, there are other ways to separate CSCs from
tumors. In 1996, Dr. Goodell observed that after adding Hoechst
33342 to a culture of bone marrow cells, a few cells did not
accumulate dyes, and he claimed that these few cells were side
population (SP) cells. Therefore, SP cells can be separated by
fluorescence screening after the outflow of Hoechst 33342.
Recently, SP cells have been identified in various normal tissues
and tumor cells. SP cells have high homology, self-renewal and
multidirectional differentiation potential.47,48 Some reports have
shown that ABCG2 is highly expressed in SP cells.47,49 ABCG2 is
highly related to the drug resistance of CSCs and is used as a
phenotypic marker for CSCs,50,51 including ovarian cancer,52
AML,53 breast cancer,54 lung cancer,55 nasopharyngeal carci-
noma,56 and hepatocellular carcinoma (HCC).57 Montanaro
et al.58 explored the optimal concentration of Hoechst 33342 to
reduce the toxic effect. The SP sorting method has universal
applicability in the separation and identification of CSCs, especially
CSCs with unknown cell surface markers, and is an effective
method for CSC research.
The colony-forming ability of CSCs is also used for separation
and identification.59 After digestion of the tumor tissues into
single cells, low-density cell culture can be conducted in serum-
free medium containing epithelial growth factor (EGF) and basic
fibroblast growth factor (FGF).60 Under this condition, a single CSC
will form a cell colony or sphere. Taylor et al.61 successfully
isolated CSCs from a variety of neurological tumors by using this
colony formation assay. However, the cell purification rate is low,
and the CSC specificity is poor in this assay. The in vivo limited
dilution assay (LDA) can be used for assessing CSC activity. After
low-density transplantation of immune-deficient mice with the
limiting dilution method, CSCs can be identified by ELDA software
analysis, and this method is affected by cell density and the
microenvironment in mice.62
Traditional chemotherapeutic drugs mainly affect cancer cells,
but CSCs are mostly arrested in the G0 phase and are relatively
static, thus evading the killing effect of chemotherapeutic drugs.63
Hence, the drug-resistant characteristics of CSCs can be used to
isolate and identify CSCs.64 Previous studies have shown that
radiotherapy combined with hypoxic culture can also be used to
enrich CSCs.65 In addition, the separation of CSCs can also be
accomplished by physical methods. Hepatoma stem cells can be
isolated from rat liver cancer tissue by Percoll density gradient
centrifugation; a cell fraction with a high nuclear-to-cytoplasmic
ratio is obtained.66 Recently, Rahimi et al.67 used the miR-302 host
gene promoter to overexpress neomycin in cancer cells and
selected and collected neomycin-resistant CSCs.
FACTORS REGULATING CSCS
CSCs can originate from at least four cell types, including normal
stem cells, directed group progenitor cells, mature cells, and the
fusion of stem cells and other mutant cells.68 Therefore,
transformed CSCs from normal cells require multiple gene
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Table 1. Various biomarkers of cancer stem cells in human cancers

Cancers Markers Function

Breast CD29+658, ALDH: An enzyme that plays a role in cell resistance665

CD49f+659, CD44: A glycoprotein involves in cell migration and self-renewal666
CD90+660, CD90: A glycoprotein participates in T cell adhesion and signal
CD133+661, transduction667
ALDH+662, CD133: A transmembrane glycoprotein that maintains lipid composition in
ESA+/CD44+/CD24,663 cell membranes668
CD44+/CD24−664 CD24: A marker that promotes blood flow in the tumor during metastasis669
CD49f: A membrane proteins of the integrin family that plays an important
role in cell surface adhesion and signaling670
Prostate EpCAM+671, α2β1: A receptor involves in cell adhesion and recognition31
CD117+672, E-cadherin: It plays an important role in tumor migration and invasion676
α2β1+31, CXCR4: CXC chemokine receptor works with CD4 protein to support HIV
ALDH+42, entry into cells675
CD44+673, EZH2: A member of the Polycomb family plays an vital role in the central
EZH2+674, nervous system674
CXCR4+675,
E-cadherin+676,
CD133+677
Brain CD49f+678, CD36: The main glycoprotein on the surface of platelet has an important
CD90+679, function as an adhesion molecule686
CD44+680, EGFR: It binds to epidermal growth factor and promote proliferative
CD36+681, migration in tumors682
EGFR+682, A2B5: A ganglioside marker that identifies subpopulations of nerve cells in
A2B5+683, the central nervous system687
L1CAM+684, L1CAM: A adhesion molecule that plays an important role in the
CD133+41,685 development of the nervous system include neuronal migration and
differentiation684
Stomach ALDH+688, CD44+689, CD44V8–10: A variant of CD44 with a specific class of CSCs690
CD44V8–10+690, CD54: A class of adhesion molecules express in malignant tumor cells693
CD133+691, CD24+692,
CD54+693, CD90+694,
CD49f+678 CD71+695,
EpCAM+696
Colorectal CD200+697, EpCAM+698, CD200: A glycoprotein plays an important role in the regulation of
CD133+699, CD166+, immunosuppression and anti-tumor activity703
CD206+700, CD44+701, CD166: It binds to the T cell differentiation antigen CD6 and involves in cell
CD49f+678, ALDH+702 adhesion and migration processes704
CD206: A mannose receptor involves in endocytosis, phagocytosis, and
immune homeostasis700
EpCAM: It expresses on most normal epithelial cells and gastrointestinal
cancers, and acts as a homotypic calcium-independent cell adhesion
molecule705
Liver CD24+706, CD133+707, CD13: A receptor for human coronavirus strains, which is the main cause of
CD13+708, CD44+709, upper respiratory tract infection and leukemia712
CD206+700, OV-6+708, OV-6: A marker for rat oval cells and hepatic stem cells708
CD90+710, EpCAM+711
AML CD34+, CD34: It plays a role in the attachment of stem cells to bone marrow
CD38−, extracellular or stromal cells713
CD90+, CD38: An intracellular Ca2+ mobilization messenger, prognostic markers for
CD71+, patients with chronic lymphocytic leukemia714
CD19+, CD71: A transferrin receptor is important for nerve development715
CD20+, CD19: A class of signal transduction molecules regulate B lymphocyte
CD44+, differentiation716
CD10+, CD20: The protein plays a role in the development and differentiation of B
CD45RA+, cells into plasma cells717
CD123+15 CD10: It inhibits a variety of peptide hormones, include glucagon,
encephalin, oxytocin, and bradykinin718
CD45RA: A class of leukocyte activation regulators719
CD123: An interleukin-specific subunit of a heterodimeric cytokine
receptor720
Melanoma CD20+721, CD271+,722, CD271: A nerve growth factor receptor mediates cell survival and cell death
ALDH+723, CD133+724 in nerve cells725
Bladder CD44v6+726, CD44+727, CD44v6: It involves in cell migration, cell adhesion729
ALDH+728
Ovarian CD24+730, ALDH+,731, CD117: A class of transmembrane receptors is also known as stem cell
CD44+/CD117+732, factors735
EpCAM+733, CD133+734

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Yang et al.
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Table 1 continued
Cancers Markers Function

Pancreas ALDH+736, CD133+30, ABCG2: A class of membrane proteins belongs to the ABC transporter
CD44+/CD24+/EpCAM+17, superfamily that plays a role in the drug resistance properties of CSCs
ABCG2+737, CXCR4+,738
HNSCC ALDH+739, CD44+,740,
CD166+741
Gallbladder CD44+/CD133+742
RCC CD133+743, ALDH+,743, CD105: TGF receptor that involves in TGF-β signaling plays a role in
CXCR4+743, CD44+,744, angiogenesis745
CD105+23
Lung CD166+746, CD90+,747, CD87: A receptor for urokinase plasminogen activator that affects many
CD87+748, ALDH+,749, normal and pathological processes associates with cell surface
CD44+750, CD133+751 plasminogen activation and local degradation of extracellular matrices748
Malignant CD9+, CD9: A glycoprotein plays a role in many cellular processes, includes
mesothelioma CD24+, differentiation, adhesion and signal transduction, and plays a key role in
CD26+752 cancer cell movement and metastasis753
CD26: A class of serine exopeptidases is also an intrinsic membrane
glycoprotein754
OSCC CD44+/CD24,−755 ITGA7: A integrin plays a role in cell migration, morphogenesis,
ITGA7+756 differentiation, and metastasis and participates in the process of
differentiation and migration during myogenesis757
cSCC CD44+758, CD133+759
Esophageal ITGA7+, CD44+,
ALDH+, CD133+,
CD90+297
MM CD138−, CD138: A member of the Syndecan proteoglycan family that involves in cell
CD19+, proliferation, cell migration, and cell–matrix interactions762
CD27+760,761 CD27: A transmembrane glycoprotein involves in the regulation of B cell
activation and immunoglobulin synthesis763
Cervix ABCG2+, CD133+, CD49f+764, ALDH+765
Nasopharyngeal CD44+766, CD133+767,
ALDH+768, CD24+769
Laryngeal ALDH+, CD44+770, CD133+771
AML acute myeloid leukemia, HNSCC head and neck squamous cell carcinoma, RCC renal cell carcinoma, OSCC oral squamous cell carcinoma, cSCC cutaneous
squamous cell carcinoma, MM multiple myeloma, ALDH aldehyde dehydrogenase, EpCAM epithelial cellular adhesion molecule

mutations, epigenetic changes, uncontrolled signaling path-
ways, and continuous regulation of the microenvironment. It is
currently believed that there are many similarities between CSCs
and embryonic stem (ES) cells, especially regarding their ability
to grow indefinitely and self-renew, signaling pathways and
some transcription factors. In addition, CSCs exist in the
supporting microenvironment, which is vital for their survival.
Moreover, the complex interaction between CSCs and their
microenvironment can further regulate CSC growth. This section
will discuss the effects of transcription factors, signaling path-
ways, and the microenvironment on CSC survival, apoptosis, and
metastasis.
Major transcription factors in CSCs
Generally, stem cells have at least two common characteristics: the
ability to self-renew and the potential to differentiate into one or
more specialized cell types.69 Somatic cells can be reprogrammed
to become induced pluripotent stem cells by transient ectopic
overexpression of the transcription factors Oct4, Sox2, Nanog,
KLF4, and MYC.70–72 In addition, there are some similarities
between CSCs and ES cells. It is reasonable that some embryonic
transcription factors can be re-expressed or reactivated in CSCs.69
Therefore, these transcription factors play a very important role in
the regulation of CSC growth.
Oct4, a homeodomain transcription factor of the Pit-Oct-Unc
family, is recognized as one of the most important transcription
factors.73 Recently, Oct4 has emerged as a master regulator that
controls pluripotency, self-renewal, and maintenance of stem
cells.74 Some studies have reported that Oct4 is highly
expressed in CSCs.70,73 High expression of Oct4 is positively
correlated with glioma grades75 and promotes self-renewal,
chemoresistance, and tumorigenicity of HCC stem cells.76 High
expression of Oct4 is also observed in breast CSC-like cells
(CD44+/CD24−).77 Cisplatin, etoposide, adriamycin, and pacli-
taxel γ-irradiation upregulate the expression of Oct4 in lung
cancer cells, and CD133+ cells are more resistant to drug
treatments than CD133− cells.78 Data also show that Oct4
expression is associated with poor clinical outcome in hormone
receptor-positive breast cancer.79 Knockdown of Oct4 also
reduces the stemness of germ cell tumors.80 Hence, these
studies have proven that Oct4 is a pluripotent factor in CSCs.
Sox2 belongs to the family of high-mobility group transcription
factors and plays a significant function in the early development
and maintenance of undifferentiated ESCs. It is also one of the key
transcription factors in CSCs. Rodriguez-Pinilla et al.81 found that
increased expression of Sox2 in basal-like breast cancer may help
to characterize poorly differentiated/stem cell phenotypes.82
Hagerstrand et al.82 also found that a high level of Sox2 can
induce xenograft glioma. Further studies showed that knockout of
Sox2 inhibits glioblastoma cell proliferation and tumorigenicity,
which suggests that Sox2 is the basis for maintaining the self-
renewal ability of tumor-initiating cells (TICs).83 Sox2 also

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maintains the self-renewal of TICs in osteosarcomas, and down-
regulation of Sox2 drastically decreases its transformative
characteristics and tumorigenesis ability in vitro. Furthermore,
osteosarcoma cells that lose Sox2 cannot form osteospheres and
differentiate into mature osteoblasts any longer.84 Sox2 is found in
invasive cutaneous squamous cell carcinoma (SCC) and promotes
the metastasis of cancer cells.85 These studies suggest that Sox2
promotes self-renewal and tumorigenesis and inhibits differentia-
tion in CSCs.
Nanog, a differentiated homeobox (HOX) domain protein that
was first discovered in ESCs, has typical self-renewal and multi-
potent transcriptional regulatory functions.86 Although Nanog is
silenced in normal somatic cells, abnormal expression has been
reported in human cancers, such as breast cancer, cervical cancer,
brain cancer, colon cancer, head and neck cancer, lung cancer,
and gastric cancer.86–90 Compared to levels in benign tissues,
Nanog messenger RNA (mRNA) is elevated in malignant tumors. In
a number of patients with colorectal cancer (n = 175), high Nanog
protein is associated with lymph node positivity and Dukes
grade.91 Similarly, overexpression of Nanog in colorectal CSCs
promotes colony formation and tumorigenicity in vivo.92 In
addition, gastric cancer patients with high Nanog levels have a
lower 5-year survival rate.88 The expression level of Nanog is
increased in HCC cell lines and primary tumors and is associated
with advanced diseases (tumor node metastasis (TNM) stage III/
IV).93 Through the study of prostatic cell lines, xenografts and
primary tumors, it was found that Nanog short hairpin RNA
inhibits the formation of primary prostate cancer cells (PCA)
spheres, clonal growth, and tumorigenesis.94 In 43 cases of
pancreatic cancer tissue microarray analysis, Kaplan–Meier analy-
sis showed that high expression of Nanog (and Oct4) predicted
worse prognosis and was negatively correlated with patient
survival.95 These studies indicate that Nanog plays an important
role in regulating the self-renewal and proliferation of CSCs.
KLF4 is expressed in many tissues and plays an important role in
many different physiological processes. As a bifunctional tran-
scription factor, KLF4 activates or inhibits transcription according
to different target genes and utilizing different mechanisms. KLF4
can play an oncogenic or anticancer role, depending on the type
of cancer involved. For example, KLF4 is an anticancer factor in the
intestinal epithelium and gastric epithelium.96 The expression of
KLF4 is downregulated with hypermethylation and loss of
heterozygosity in colorectal CSCs and gastric CSCs.97 Down-
regulation of KLF4 is also found in other cancers, such as non-
small-cell lung carcinoma,98 liver cancer,99 leukemia,100 anaplastic
meningioma,101 bladder cancer,102 and esophageal cancer.103
Although these data clearly demonstrate that KLF4 plays an
anticancer role in those cancers, KLF4 may also be an oncogene,
which was demonstrated for the first time in nearly a decade.104
Overexpression of KLF4 in transformed rat renal epithelial cells
induces tumorigenesis of laryngeal SCC.105 In addition, depletion
of KLF4 inhibits melanoma xenograft growth in vivo.106 High
expression of KLF4, an oncogene in human breast CSCs, is
correlated with an aggressive phenotype in canine mammary
tumors.107 These studies suggest that KLF4 has different functions
in different CSCs.
MYC has three family members (C-Myc, N-Myc, and L-Myc,
which are encoded by the proto-oncogene family and are
essential transcription factors in the DNA-binding proteins of the
basic helix–loop–helix (bHLH) superfamily). MYC regulates a large
number of protein-coding and noncoding genes and coordinates
various biological processes in stem cells, such as cell metabolism,
self-renewal, differentiation, and growth.108,109 Although the MYC
gene is one of the most commonly activated oncogenes that is
involved in the pathogenesis of human cancer, overexpression of
MYC alone is surprisingly unable to induce the transformation of
normal cells into tumor cells. The overexpression of MYC in normal
human cells may be ineffective or highly destructive, resulting in
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Yang et al.
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stagnation of proliferation, aging, or apoptosis.110 MYC is usually
deregulated in human cancers, plays an important role in
maintaining the number of invasive CSCs,111 and is also one of
the most effective oncogenes for detecting the cell transformation
phenotype in vitro and in vivo. Previous studies have shown that
deletion of the tumor suppressor gene p53 and MYC synergizes to
induce hepatocyte proliferation and tumorigenesis.112 In addition
to p53 deletion, overexpression of Bcl-2 and Bmi-1 and loss of
p19ARF also assist MYC in regulating the survival and proliferation
of CSCs.113 The expression of the three members of the MYC
family is different in different tumors, such as C-MYC in leukemia
and tongue SCC stem cells114,115 and N-MYC in small-cell lung
cancer, prostate cancer, neuroblastoma, and medulloblas-
toma.116,117 L-MYC is expressed in hematopoietic malignancies.118
In addition, inactivation of MYC results in HCC stem cells
differentiating into hepatocytes and biliary duct cells to form bile
duct structures, which might be associated with the loss of the
tumor marker α-fetoprotein and increased expression of cytoker-
atin 8, hepatocyte markers, carcinoembryonic antigen, and the
liver stem cell marker cytokeratin 19.119 Studies have also shown
that MYC is highly expressed in glioblastoma multiforme stem
cells and induces cell proliferation and invasion and inhibits
apoptosis.111 Increased copy number of the MYC gene in human
and mouse prostate CSCs has also been found.120 These studies
indicate that MYC induces tumorigenesis with the help of other
factors.
Major signaling pathways in CSCs
Many signaling pathways that contribute to the survival,
proliferation, self-renewal, and differentiation properties of normal
stem cells are abnormally activated or repressed in tumorigenesis
or CSCs. Many endogenous or exogenous genes and microRNAs
regulate these complex pathways. These signaling pathways can
also induce downstream gene expression, such as cytokines,
growth factors, apoptosis genes, antiapoptotic genes, proliferation
genes, and metastasis genes in CSCs. These signaling pathways
are not a single regulator but interwoven networks of signaling
mediators to regulate CSC growth. Therefore, this section will
describe how signaling pathways regulate CSC growth.
Wnt signaling pathway in CSCs. Wnts include large protein
ligands that affect diverse processes, such as the generation of cell
polarity, and cells fate.121 The Wnt pathway is highly complex and
evolutionarily conserved and includes 19 Wnt ligands and more
than 15 receptors.122 The Wnt signaling pathway can be divided
into canonical Wnt signaling (through the FZD-LRP5/6 receptor
complex, leading to derepression of β-catenin) and noncanonical
Wnt signaling (through FZD receptors and/or ROR1/ROR2/RYK
coreceptors, activating PCP, RTK, or Ca2+ signaling cascades).123 In
canonical Wnt signaling, in the absence of Wnt ligands (inactive
Wnt signaling state, Fig. 1, left), β-catenin is phosphorylated by
glycogen synthase kinase 3β (GSK3β), which leads to β-catenin
degradation via β-TrCP200 ubiquitination and inhibits transloca-
tion of β-catenin from the cytoplasm to the nucleus.124 In contrast,
in the presence of Wnt ligands (e.g., Wnt3a and Wnt1), the ligands
combine with Fzd receptors and LRP coreceptors (active Wnt
signaling, Fig. 1, right). LRP receptors are phosphorylated by
GSK3β and CK1α.125 β-Catenin is released from the Axin complex
to enter the nucleus. In addition, β-catenin combines with LEF/TCF
and enhances the recruitment of histone-modifying coactivators,
such as BCL9, Pygo, CBP/p300, and BRG1, to activate transcription.
Noncanonical Wnt signaling does not involve β-catenin. During
Wnt/PCP signaling, Dvl is activated through binding of Wnt
ligands and the ROR-Frizzled receptor.126 Dvl inhibits the binding
of the small GTPase Rho and the cytoplasmic protein DAAM1.127
The small GTPases Rac1 and Rho together trigger ROCK (Rho
kinase) and JNK (c-Jun N-terminal kinase). This results in
cytoskeletal rearrangement and/or transcriptional responses.128
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Fig. 1 Wnt/β-catenin pathway in cancer stem cells. The canonical Wnt/β-catenin pathway regulates the pluripotency of CSCs and determines
the differentiation fate of CSCs. In the absence of Wnt signaling, β-catenin is bound to the Axin complex, which contains APC and GSK3β, and
is phosphorylated, leading to ubiquitination and proteasomal degradation through the β-Trcp pathway. However, the complex (TAZ/YAP), the
long noncoding RNA TIC1 and proteins (TRAP1 and TIAM1) regulate the β-Trcp pathway. In the presence of Wnt signaling, the binding of
LRP5/6 and Fzd inhibits the activity of the Axin complex and the phosphorylation of β-catenin, which makes β-catenin enter the nucleus, and
then bind to TEF/TCF to form a complex, which then recruits cofactors to initiate downstream gene expression. Some proteins (DKK2
(Dickkopf-related protein 2), DACT1, CDH11, GECG, PKM2, EZH2, CD44v6, MYC, and TERT), microRNAs (miR-1246, miR-9, miR-92a, miR-544a,
and miR-483-5p), and long noncoding RNAs (lncR-β-catm and lncR-TCF7) regulate the activation of the Wnt/β-catenin pathway in CSCs

Wnt/Ca2+ signaling is activated by G protein-triggered phospho-
lipase C activity, which results in intracellular calcium flux and
downstream calcium-dependent cytoskeletal and/or transcrip-
tional responses.129,130
Aberrant Wnt signaling is found in many cancers, such as
invasive ductal breast carcinomas,131 colorectal cancer,132 papil-
lary thyroid cancer,133 esophageal cancer,134 and colorectal
cancer.135 The activation of Wnt signaling is different in different
tumors. Some Wnt activation is caused by mutations in Wnt
components, such as Axin mutation in gastrointestinal cancers,136
APC mutation in colorectal cancer,137 and β-catenin mutation in
gastric cancer and liver cancer.138,139 GSK3 genes are critical for
β-catenin regulation; therefore, many researchers expect the
occurrence of GSK3 mutations, but GSK3 mutations are not
correlated with cancer occurrence. In addition, some genes
(pyruvate kinase isozyme M2 (PKM2) in breast cancer140 and
telomerase reverse transcriptase (TERT) in prostate cancer141) and
microRNAs (miR-164a in colorectal cancer142 and miR-582-3p in
non-small-cell lung cancer143) inhibit the activity of APC, Axin, and
GSK3β to promote the accumulation of β-catenin in the
cytoplasm.
Stem cell signaling pathways and transcriptional circuits are
related to the alteration or reactivation of signaling pathways.144
Tumor dormancy is a lag phenomenon in tumor growth.
Dormancy may occur during primary tumor formation or in the
diffusion of some of the constituent tumor cells. However, primary
tumor dormancy and metastatic dormancy seem to be different
processes.145 In some cases, cells in the TME produce cytokines,
such as Wnt proteins, secreted inhibitors of bone morphogenetic
protein (BMP), and Delta, which activate the signaling pathway to
maintain the self-renewal ability of CSCs.146 Activation of Wnt
induce the transformation of dormant CSCs into active CSCs to
promote cell cycle progression through β-catenin, increasing the
expression of downstream cyclin D1 and MYC, and MYC also
promotes the expression of the polycomb repressor complex 1
component Bmi-1 and induces the combination E2F with cyclin
E.147 The extracellular matrix (ECM) protein tenascin C often exists
in the gap of stem cells, which supports the cell cycle in breast
cancer cells by increasing Wnt signals.148 In addition, aberrant Wnt
signaling has also been observed in the self-renewal of CSCs (Fig. 1).
Many reports have proven that numerous proto-oncogenes
stimulate this process through the Wnt signaling pathway.135
PKM2 catalyzes the last step of glycolysis and plays an essential
role in the proliferation of breast CSCs by associating with
increased β-catenin levels at regions “−410 to 180 and −2250 to
2000”.140,145,149 Enhancer of zeste homolog 2 (EZH2), a key
component of the polycomb PRC2 complex, promotes self-
renewal of CSCs by activating β-catenin.150 Moreover, TERT, an
RNA-dependent DNA polymerase, acts as a cofactor and forms a
complex with β-catenin to activate Wnt downstream targets in

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prostate CSCs.141 Capillary morphogenesis gene 2 increases the
expression of nuclear β-catenin to regulate the self-renewal and
tumorigenicity of gastric CSCs,151 and SMYD3, which is located
downstream of the Wnt pathway, has a similar effect.152 In
addition, long noncoding RNAs and microRNAs also promote self-
renewal of CSCs through the Wnt signaling pathway. LncTCF7
recruits the SWI/SNF complex to regulate the expression of the
TCF7 promoter in liver CSCs.153 Lnc-β-Catm associates with the
methyltransferase EZH2 to suppress the ubiquitination of
β-catenin and promote its stability,154 and LncTIC1 interacts with
β-catenin and maintains its stability, activating Wnt/β-catenin
signaling.155 MicroRNA-1246, miR-19, and miR-92a suppress the
expression of AXIN and GSK3β in CSCs.156 MicroRNA-544a
downregulates GSK3β in lung CSCs.157 MicroRNA-483-5p upregu-
lates the expression of β-catenin in gastric CSCs.158 In addition,
there are still many genes, microRNAs, and noncoding RNAs in
CSCs’ self-renewal through the Wnt signaling pathway.
Wnt signaling also plays an important role in the dedifferentia-
tion of CSCs. HOXA5, which is a member of the HOX family,
induces the differentiation of colorectal CSCs. However, Wnt
indirectly suppresses indirectly via MYC, which is an important
direct target of β-catenin/TCF in the intestine.159 PMP22, an
integral membrane glycoprotein in myelin in the peripheral
nervous system, induces the differentiation of gastric CSCs, but its
mRNA level declines with activation of the Wnt/β-catenin path-
way.160 Moreover, TRAP1, a component of the HSP90 (heat-shock
protein 90) chaperone family, inhibits the differentiation of
colorectal carcinoma stem cells by modulating β-catenin ubiqui-
tination and phosphorylation.161 Lgr5, a member of the G protein-
coupled receptor (GPCR) family of proteins, is located downstream
of the Wnt signaling pathway and restrains the differentiation of
esophageal SCC stem cells.162
Wnt signaling also plays an important role in regulating CSC
apoptosis. Dickkopf-related protein 2 induces G0/G1 arrest and
cell apoptosis by suppressing β-catenin activity in breast CSCs.163
DACT1, a homolog of Dapper that is located at chromosomal
region 14q23.1, promotes apoptosis in breast CSCs by antagoniz-
ing the Wnt/β-catenin signaling pathway.164 Cadherin-11, a
proapoptotic tumor suppressor, reduces the level of active
phospho-β-catenin (ser552) to induce apoptosis in colorectal
CSCs.165 Epigallocatechin-3-gallate increases apoptosis by degrad-
ing β-catenin in lung CSCs.166 The small-molecule inhibitor
CWP232228 antagonizes the binding of β-catenin to TCF in the
nucleus to induce apoptosis in liver CSCs.167 In addition,
temozolomide combined with miR-125b significantly induces
apoptosis by targeting the Wnt/β-catenin signaling pathway in
glioma stem cells.168
Wnt/β-catenin signaling has been implicated in CSC-mediated
metastasis.169 In the cytomembrane, Frizzled8 promotes bone
metastasis in prostate CSCs.170 The leucine-rich repeat containing
GPCR4 (LGR4, or GPR48), together with its family members LGR5/
6, binds to R-spondins 1–4 and leads to Wnt3A potentiation,
activating Wnt signaling in breast CSCs.171,172 Increased levels of
CD44v6 mRNA in human pancreatic CSCs, lung CSCs, and colon
CSCs promote migration and metastasis through the activation of
β-catenin.173–175 In the cytoplasm, TAZ/YAP interacts directly with
β-catenin and restricts β-catenin degradation,176 but TIAM1
antagonizes TAZ/YAP accumulation and translocation from the
cytoplasm to the nucleus.177 Moreover, CDH11 inhibits the
migration and invasion of colorectal CSCs by inhibiting Wnt/
β-catenin and AKT/RhoA signaling.165 Wnt signaling decreases the
expression of HOXA5 to promote CSC metastasis.159 These data
suggest that amplified Wnt signaling is important for self-renewal,
dedifferentiation, apoptosis inhibition, and metastasis of CSCs.
Notch signaling pathway in CSCs. The Notch signaling pathway
consists of the Notch receptor, Notch ligand (DSL protein), CSL
(CBF-1, suppressor of hairless, Lag), DNA-binding protein, other
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effectors, and Notch regulatory molecules. In 1917, studies
discovered the Notch gene in a mutant Drosophila. Mammals
have four Notch receptors (Notch1–4) and five Notch ligands
(Delta-like 1, 3, and 4, Jagged 1, and Jagged 2).178 Notch and DSL
ligands are transmembrane proteins that mediate communication
between neighboring cells. Under physiological conditions, the
ligand binds to a Notch receptor that is expressed on neighboring
cells in a juxtacrine manner, thereby triggering proteolytic
cleavage of the intracellular domain (ICD) of Notch and its
translocation into the nucleus to bind to the transcription factor
CSL, forming the NICD/CSL transcriptional activation complex,
which activates target genes of the bHLH transcription inhibitor
family, such as HES, HEY, and HERP.179,180
The Notch pathway regulates cancer cells in many tumors, such
as glioblastoma, leukemia, and those of the breast, pancreas,
colon, and lung, among others.181 Different tumors and tumor
subtypes express different Notch ligands and receptors. Therefore,
Notch is known to function as both an oncogene and a
suppressive gene. As an oncogene, Notch is overexpressed in
gastric cancer,182 breast cancer,183 colon cancer,184 and pancreatic
cancer. In contrast, Notch expression is downregulated in prostate
cancer,185 skin cancer,186 non-small-cell lung cancer,187 liver
cancer,188 and some breast cancers.189 Whether Notch acts as
an oncogene or a tumor suppressor gene is determined by the
microenvironment.190 Moreover, post-translational modifications
of Notch receptors change their affinity for ligands and their
intracellular half-lives.191
Many studies on the Notch pathway in CSCs have shown that
activation of Notch promotes cell survival, self-renewal, and
metastasis and inhibits apoptosis. Aberrant Notch signaling
(Notch1 and Notch4) promotes self-renewal and metastasis of
breast and HCC stem cells.192,193 However, microRNA-34a down-
regulates Notch1.194 Similarly, abundant Delta-like ligand 4 (DLL4)
also promotes tumor angiogenesis and metastasis in gastric
CSCs.195 Delta-like 1 activation of Notch1 signaling requires the
assistance of the actin-related protein 2/3 complex to maintain the
stem cell phenotype of glioma-initiating cells.196 Additionally,
some intracellular genes also regulate the Notch signaling
pathway. For example, MAP17 (DD96, PDZKIP1), a nonglycosylated
membrane-associated protein, is located on the plasma mem-
brane and the Golgi apparatus. MAP17 interacts with NUMB
through the PDZ-binding domain to activate the Notch pathway
in cervical CSCs.197 Inducible nitric oxide synthase promotes the
self-renewal capacity of CD24+CD133+ liver CSCs through TACE/
ADAM17 activation to regulate Notch1 signaling.198 Moreover,
tumor necrosis factor-α (TNFα) enhances the CSC-like phenotype
by activating Notch1 signaling in oral SCC cells.199 Overexpression
of PER3 decreases the expression of Notch1 and Jagged 1 in
colorectal CSCs.200 In addition, KLF4 and BMP4 also increase
Notch1 and Jagged 1 in breast CSCs to regulate cell migration and
invasion.201,202 BRCA1 is a key regulator of breast cancer cell
differentiation; however, it is localized to a conserved intronic
enhancer region within the Notch ligand Jagged 1 gene to
maintain the stemness of breast CSCs.203 Similarly, increased Gli3
also promotes cell proliferation and invasion in oral SCC by
increasing Notch2.204 Hypoxia/hypoxia-inducible factor (HIF)-
induced migration and invasion is a well-known phenomenon
that has been reported in numerous CSCs.205 Notch1 can induce
the migration and invasion of ovarian CSCs in the absence of
hypoxia.206 Hypoxia-induced Jagged 2 activation enhances cell
invasion of breast CSCs207 and lung CSCs.208 Moreover, HIF-1α/2α
regulates self-renewal and maintenance of glioblastoma stem
cells.209 In addition, increased miR-200b-3p decreases Notch
signaling to promote pancreatic CSCs to become asymmetric.210
MiR-26a directly targets Jagged 1 to inhibit osteosarcoma CSC
proliferation.211 These studies indicate that Notch plays an
important role in regulating the self-renewal, growth, and
metastasis of CSCs.
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Hh signaling pathway in CSCs. The Hh signaling pathway consists
of ligands and receptors. The Hh signaling network is very
complex, including extracellular Hh ligands, the transmembrane
protein receptor PTCH, the transmembrane protein SMO, inter-
mediate transduction molecules, and the downstream molecule
GLI.212 The components of the Hh signaling pathway play different
roles. The membrane protein SMO plays a positive regulatory role,
while the transmembrane protein PTCH plays a negative
regulatory role. PTCH has two subtypes, PTCH1 and PTCH2,213
and there is 73% homology between the two subtypes. GLI, an
effector protein, has three subtypes, Gli1, Gli2, and Gli3, in
vertebrates,214 and these effector proteins have different func-
tions. Gli1 strongly activates transcription, while Gli3 inhibits
transcription.215 Gli2 has dual functions of activating and
inhibiting transcription but mainly functions as a transcriptional
activator.216,217 Numerous studies have confirmed that Hh
signaling is involved in embryonic development and the
formation of the nervous system, skeleton, limbs, lung, heart,
and gut.218 As an extracellular signaling pathway, in the presence
of ligand signals, Hh ligands bind to PTCH receptors on target cell
membranes and initiate a series of intracellular signal transduction
processes.219 When there is no ligand signal, the transmembrane
receptor PTCH on the target cell membrane binds to SMO and
inhibits SMO activity, which prevents signaling.220 When the Hh
ligand is present, it binds to PTCH, which changes the spatial
conformation of PTCH, removing the inhibition of SMO activating
the transcription factor GLI and inducing it to enter the cell
nucleus, where GLI regulates cell growth, proliferation, and
differentiation.221
Studies have confirmed that abnormal activation of the Hh
signaling pathway can be found in human cancers,222 such as
breast cancer,223 lung cancer,224 bladder cancer,225 pancreatic
cancer,226 chondrosarcoma,227 rhabdomyosarcoma,228 neuroblas-
toma,229 medulloblastoma,230 and gastric cancer.231 However,
activation of Hh signaling is different in different tumors. Gorlin
syndrome (basal cell nevus syndrome), an autosomal dominant
condition, is associated with germline loss of the PTCH1 gene. This
condition is very common in basal cell carcinoma, rhabdomyo-
sarcoma, and medulloblastoma.232,233 Other Hh pathway compo-
nents are also mutated in human cancers, such as Gli1 and Gli3
mutations in pancreatic adenocarcinoma, Gli1 gene amplification
in glioblastoma, and SUFU (suppressor of fused) mutations in
medulloblastoma.234,235 In addition, other genes also regulate the
Hh signaling pathway. Speckle-type POZ protein, an E3 ubiquitin
ligase adaptor, inhibits Hh signaling by accelerating Gli2
degradation in gastric cancer.236
Hh signaling plays distinct functions in different types of
cancer.237 During tumor development, Hh signaling has three
major roles: driving tumor development, promoting tumor
growth, and regulating residual cancer cells after therapy. Based
on these functions, the aberrant Hh pathway plays a causal role in
CSCs238,239 (Fig. 2). The expression level of Hh signaling

Fig. 2 Hedgehog signaling pathway in cancer stem cells. The Hedgehog pathway plays a key role in stem maintenance, self-renewal, and
regeneration of CSCs. The secreted Hh protein acts in a concentration- and time-dependent manner to initiate a series of cell responses, such as
cell survival, proliferation, and differentiation. After receiving the Shh signal, the transmembrane protein receptor PTCH relieves the inhibition of
the transmembrane protein SMO, which induces Gli1/2 to detach from SUFU and enter the nucleus to regulate downstream gene transcription.
During activation of the Hh pathway, some proteins (IL-6, IL-27, Fbxl17 (F-box and leucine-rich repeat protein 17), PPKCI, RARα2, RUXN3, SCUBE2,
HDAC6 (histone deacetylase 6), USP48, CK2α, WIP1, GALNT1, VASH2 (Vasohibin 2), BCL6, FOXC1 (forkhead box C1), and p65), microRNAs (miR-324-
5p, miR-122, and miR-326), and the long noncoding RNA HDAC2 are involved in the Hedgehog pathway to affect CSC growth

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components is relatively high in CSCs. For example, Hh signaling
promotes the maintenance, proliferation, self-renewal, and
tumorigenicity of lung adenocarcinoma stem cells.240 In CD133+
glioma stem cells, SMO, GLI, and PTCH promote cell proliferation,
self-renewal, migration, and invasion. The expression of Gli1,
PTCH1, and PTCH2 is regulated by histone deacetylase 6.241 USP48
activates Gli-dependent transcription by stabilizing the Gli1
protein in glioma stem cells.242 The protein kinase CK2α enhances
Gli1 expression and its transcriptional activity in lung CSCs.243
WIP1 (PPM1D), a nuclear Ser/Thr phosphatase, also enhances the
function of Gli1 by increasing its transcriptional activity, protein
stability, and nuclear localization in breast CSCs and medullo-
blastomas.244,245 F-box and leucine-rich repeat protein 17
mediates the release of Gli1 from SUFU for proper Hh signal
transduction in medulloblastoma stem cells.246 Moreover, retinoic
acid receptor α2 (RARα2) upregulates the expression of SMO and
Gli1 in CD138+ multiple myeloma stem cells.247 PRKCI, which is
regulated by miR-219 in tongue SCC,248 has a similar function as
RARα2 in maintaining a stem-like phenotype in lung SCC cells.249
Interleukin-27 (IL-27) and IL-6 activate Hh signaling in CD133+
non-small-cell lung CSCs.250 During self-renewal and maintenance
of stemness of BCMab1+CD44+ bladder CSCs, glycotransferase
GALNT1-mediated glycosylation significantly activates Sonic Hh
signaling by upregulating Gli1.251
Furthermore, p63, a master regulator of normal epithelial stem
cell maintenance, regulates the expression of Shh, Gli2, and
PTCH1 by directly binding to their gene regulatory regions, which
eventually contributes to the activation of Hh signaling in
mammary CSCs.252 The N-terminal domain of forkhead box C1
binds directly to an internal region (amino acids (aa) 898–1168) of
Gli2 to enhance transcriptional activation of Gli2 and determines
the stem cell phenotype in breast CSCs.253 Through recruitment
of the deubiquitinating enzyme ATXN3, tetraspanin-8 interacts
with PTCH1 and inhibits the degradation of the SHH/PTCH1
complex. In addition, long noncoding microRNAs also activate Hh
signaling. For example, lncHDAC2 promotes the self-renewal of
liver CSCs by recruiting the NuRD complex onto the promoter of
the PTCH1 gene to suppress its expression.254 In addition, the TME
is crucial for the survival of CSCs. Consequently, breast CSCs
secrete Shh, which upregulates cancer-associated fibroblasts
(CAFs). Subsequently, CAFs secrete factors that promote the
expansion and self-renewal of breast CSCs.255 Hh signaling also
promotes self-renewal and metastasis of CSCs by upregulating
the expression of related downstream markers of CSCs, such as
Bmi-1, Wnt2, ALDH1, CD44, CCND1, Twist1, C-MYC, Nanog, Oct4,
PDGFRα (platelet-derived factor receptor-α), Snail, Jagged 1, and
C-MET.231,247,256–264
Some proto-oncogenes and suppressor genes also directly or
indirectly regulate Hh signaling in the proliferation and migration
of CSCs. The signal peptide CUB EGF-like domain-containing
protein 2 (SCUBE2), a member of the SCUBE family of proteins,
inhibits cell proliferation and migration in glioma stem cells by
downregulating Hh signaling.265 BCL6, a transcriptional repressor
and lymphoma oncoprotein, directly represses the Sonic Hh
effectors Gli1 and Gli2 in medulloblastoma stem cells.266 The
transcription factor RUNX3 suppresses metastasis and the
stemness of colorectal CSCs by promoting ubiquitination of Gli1
at the intracellular level.267 Vasohibin 2 suppresses Smo, Gli1, and
Gli2 expression in pancreatic CSCs.268 β-Catenin stably increases
its physical interaction with Gli1, resulting in Gli1 degradation in
medulloblastoma stem cells.269 In addition, microRNAs also target
Hh signaling components to regulate CSC proliferation. For
example, miR-324-5p significantly decreases SMO and Gli1 in
myeloma stem cells.270 Mir-326 directly downregulates SMO and
Gli2 in medulloblastoma stem cells.271 MiR-326 downregulates
SMO in glioma stem cells.272 Mir-122 targets Shh and Gli1 in lung
CSCs.273 These data demonstrate that amplified Hh signaling is
important for the self-renewal, growth, and metastasis of CSCs.
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NF-κB signaling pathway in CSCs. Nuclear factor-κB (NF-κB), a
rapidly inducible transcription factor,274 consists of five different
proteins (p65, RelB, c-Rel, NF-κB1, and NF-κB2). The main
physiological function of NF-κB is the p50-p65 dimer.275–277 The
primary mode of NF-κB regulation occurs at the level of
subcellular localization. In the activation stage, transcription factor
complexes must translocate from the cytoplasm to the nucleus.278
The activity of the complexes is regulated by two major pathways
(canonical NF-κB signaling and noncanonical NF-κB signaling). In
the canonical NF-κB activation pathway, activation occurs through
the binding of ligands, such as bacterial cell components, IL-1β,
TNF-α, or lipopolysaccharides, to their respective receptors, such
as Toll-like receptors, TNF receptor (TNFR), IL-1 receptor (IL-1R),
and antigen receptors.279 Stimulation of these receptors leads to
the phosphorylation and activation of IκB kinase (IKK) proteins,
subsequently initiating the phosphorylation of IκB proteins.276 The
alternative pathway of NF-κB activation is termed the noncano-
nical pathway. The noncanonical pathway receptor originates
from different classes, such as CD40, receptor activator for NF-κB,
B cell activation factor, TNFR2 and Fn14, and lymphotoxin
β-receptor.280 This pathway leads to activation of NF-κB by
inducing the kinase (NIK), which then phosphorylates and
predominantly activates IKK1. The activity of the latter enzyme
induces the phosphorylation of p100 to generate p52.281
The NF-κB pathway plays an important role in regulating
immune and inflammatory responses. In addition, the NF-κB
pathway is involved in cellular survival, proliferation, and
differentiation.276 The process of tumor development and
progression produces cytokines, growth, and angiogenic factors
and proteases to activate NF‐κB signaling.282 Inflammation has
been recognized as a hallmark of cancer.283 Overactivation of NF-
κB signaling has been reported in gastrointestinal, genitourinary,
gynecological, and head and neck cancers, breast tumors, multiple
myeloma, and blood cancers.278,284–286 However, direct or altered
molecular mutations in NF-κB have rarely been reported in human
cancers.287 Based on recent studies, NF-κB regulates many genes
and is implicated in cell survival, proliferation, metastasis, and
tumorigenesis of cancer.288 NF-κB activation also directly or
indirectly enhances the expression of key angiogenesis factors
and adhesion molecules, such as IL-8, vascular endothelial growth
factor (VEGF), and growth-regulated oncogene 1.289
The NF-κB pathway has an essential connection regulating
inflammation, self-renewal, or maintenance and metastasis of
CSCs (Fig. 3). CD44+ cells promote self-renewal, metastasis, and
maintenance of ovarian CSCs by increasing the expression of RelA,
RelB, and IKKα and mediating nuclear activation of p50/RelA (p50/
p65) dimer.290 High levels of NIK induce activation of the
noncanonical NF-κB pathway to regulate the self-renewal and
metastasis of breast CSCs.291 Moreover, stromal cell-derived
factor-1 (SDF-1) also has the same effect by regulating the
translocation of p65 from the cytoplasm to the nucleus.292 The
inflammatory mediator prostaglandin E2 (PGE2) contributes to
tumor formation, maintenance, and metastasis by activating NF-
κB via EP4-PI3K (phosphoinositide 3-kinase) and EP4-MAPK
pathways in colorectal CSCs.293 Chemokines, low-molecular-
weight proinflammatory cytokines, are important mediators of
cell proliferation, metastasis, and apoptosis.294 C-C chemokine
receptor 7 interacts with its ligand chemokine ligand 21 to inhibit
apoptosis and induce survival and migration in CD133+ pancreatic
cancer stem-like cells by increasing the expression of extracellular
signal-regulated kinase 1/2 (Erk1/2) and p65.295 Furthermore, B
cell-specific Moloney murine leukemia virus integration site 1
(Bmi-1) also enhances the p65 protein in gastric CSCs.296
MicroRNAs also play an important role in promoting the
proliferation of CSCs. Mir-221/222 promotes self-renewal, migra-
tion, and invasion in breast CSCs by inhibiting the expression of
PTEN and then inducing the phosphorylation of AKT, resulting in
elevated p65, p-p65, and COX2.297
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Fig. 3 NF-κB signaling pathway in cancer stem cells NF-κB proteins are involved in the dimerization of transcription factors, regulate gene
expression, and affect various CSC biological processes, including inflammation, stress responses, growth, and development of CSCs. The
main physiological function of NF-κB is the p50-p65 dimer. The active p50-p65 dimer is further activated by post-translational modification
(phosphorylation, acetylation, or glycosylation) and transported into the nucleus, which induces the expression of target genes in
combination with other transcription factors. Some proteins (CD44, CD146, TNFRSF19, Bmi-1, FOXP3, and SDF-1) and microRNAs (miR-221 and
miR-222) directly regulate the NF-κB pathway. In addition, some proteins (PGE2, GIT-1 (G protein-coupled receptor kinase-interacting protein
1), C-C chemokine receptor 7 (CCR7), and TGF-β) and miR-491 indirectly affect the NF-κB pathway via the ERK and MAPK pathways in CSCs

In addition, other transcription factors also inhibit self-renewal
and metastasis in CSCs by the NF-κB pathway. Increased
expression of FOXP3 has been identified in different cancers.298
FOXP3 interacts with NF-κB, inhibits the expression of COX2
located downstream of NF-κB, and affects self-renewal and
metastasis in colorectal CSCs.299 Overexpression of miR-491 blocks
the activation of NF-κB in liver CSCs by targeting G protein-
coupled receptor kinase-interacting protein 1, which inhibits
ERKs.300 Moreover, some drugs inhibit cell proliferation and
metastasis of CSCs by the NF-κB pathway. Disulfiram, an anti-
alcoholism drug, inhibits tumor growth factor-β (TGF-β)-induced
metastasis via the ERK/NF-κB/Snail pathway in breast CSCs.301
Sulforaphane preferentially inhibits self-renewal in triple-negative
breast CSCs by inhibiting NF-κB p65 subunit translocation and
downregulating p52 and its transcriptional activity.302 Curcumin
regulates the proliferation, metastasis, and apoptosis of HCC stem
cells by inhibiting the NF-κB pathway.303 These data demonstrate
that amplified NF-κB signaling is important for regulating
apoptosis, proliferation, and metastasis of CSCs.
JAK-STAT signaling pathway. The Janus kinase/signal transducers
and activators of transcription (JAK-STAT) signaling pathway is a
signal transduction pathway that is stimulated by cytokines. This
pathway is involved in many important biological processes, such
as cell proliferation, differentiation, apoptosis, and immune
regulation. Compared with the complexity of other signaling
pathways, this signaling pathway is relatively simple. There are
three components: the tyrosine kinase-related receptor, the
tyrosine kinase JAK, and the transcription factor STAT.304 Many
cytokines and growth factors transmit signals through the JAK-
STAT signaling pathway, including interleukin-2-7, granulocyte/
macrophage colony-stimulating factor, growth hormone, EGF,
PDGF, and interferon.305 These cytokines and growth factors have
corresponding receptors on the cell membrane. The common
characteristic of these receptors is that the receptor itself does not
have kinase activity, but there is a binding site for the tyrosine
kinase JAK in the cells. After binding with ligands, tyrosine
residues of various target proteins are phosphorylated through
JAK activation to achieve signal transduction from the extracellular
to intracellular space. The JAK protein family consists of four
members: JAK1, JAK2, JAK3, and Tyk2.306 JAK proteins have seven
JAK homology (JH) domains in their structures. The JH1 domain is
the kinase domain, the JH2 domain is the "pseudo" kinase domain,
and JH6 and JH7 are the receptor binding domains.307 STAT is
called "signal transducer and activator of transcription". As the
name implies, STAT plays a key role in signal transduction and
transcriptional activation. At present, seven members of the STAT
family (STAT1, STAT2, STAT3, STAT4, STAT5a, STAT5b, STAT6) have
been identified. The structure of STAT protein can be divided into
the following functional regions: N-terminal conserved sequence,
DNA-binding region, SH3 domain, SH2 domain, and C-terminal
transcriptional activation region.308 Generally, many cytokines and
growth factors integrate with tyrosine kinase-related receptors.
After receiving the signal from the upstream receptor molecule,

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JAK is quickly recruited to and activates the receptor, resulting in
JAK activation to catalyze tyrosine phosphorylation of the
receptor. The phosphorylated tyrosine on the receptor molecule,
which is a signaling molecule, can bind with the SH2 site of
STAT.309 When STAT binds to the receptor, tyrosine phosphoryla-
tion of STAT also occurs, which forms a dimer and enters the
nucleus.310 As an active transcription factor, the STAT dimer
directly affects the expression of related genes and then changes
the proliferation or differentiation of target cells.311
Constitutive activation of JAKs and STATs was first recognized
as being associated with malignancy in the 1990s.312 Based on
current studies, JAK2 mutation and abnormal activation of STAT3
are prone to occur in many tumors.313 Mutations in JAK2 have
been reported in the majority of patients with myeloproliferative
neoplasms,314 such as polycythemia vera, myelofibrosis, and
thrombocythemia.315,316 These disorders are caused by the
overexpansion of hematopoietic precursors, which are often
clonal and can result in leukemia.314 Several lines of evidence
show that constitutive activation of JAK2 and STAT3 in the
absence of any stimulating ligand occurs in polycythemia
vera.317,318 Moreover, studies have also found aberrant activation
of STATs in human cancers, such as head and neck cancer,319
endometrial cancer,320 breast cancer, diffuse large B cell
lymphoma,321 HCC,322 colorectal cancer, glioma,323 and colon
cancer.324 Furthermore, aberrant STAT5 signaling has been found
in the pathogenesis of hematologic and solid organ malignan-
cies.325,326
The JAK/STAT pathway is evolutionarily conserved. This path-
way promotes the survival, self-renewal, hematopoiesis, and
neurogenesis of ESCs.327 This pathway is also activated in CSCs.
The persistent activation of STAT3 significantly promotes cell
survival and the maintenance of stemness in breast CSCs.328 IL-10
induces cell self-renewal, migration, and invasion in non-small-cell
lung CSCs.329 IL-6 activates the JAK1/STAT3 pathway in ALDHhigh
CD126+ endometrial CSCs.320 Furthermore, IL-6 also induces the
conversion of nonstem cancer cells into cancer stem-like cells in
breast cancer by the activating downstream Oct4 gene.330 Oct4
also activates the JAK1/STAT6 pathway in ovarian CSCs.331 In
CD44+CD24− breast and colorectal CSCs, erythropoietin, and IL-6
activate the JAK2/STAT3 pathway.332–334 Retinol-binding protein 4
activates JAK2/STAT3 signaling by its STRA6 receptor in colon
CSCs.319 HIF-1α enhances the self-renewal of glioma stem-like cells
by the JAK1/STAT3 pathway.335 AJUBA is a scaffold protein that
participates in the regulation of cell adhesion, differentiation,
proliferation, and migration and promotes the survival and
proliferation of colorectal CSCs via the JAK1/STAT1 pathway.336
Moreover, microRNAs are also involved in activating JAK/STAT
signaling by inhibiting negative regulatory factors of JAK2/STAT3.
For example, miR-500a-3p targets multiple negative regulators of
the JAK2/STAT3 signaling pathway, such as SOCS2, SOCS4, and
PTPN, in HCC stem cells, leading to constitutive activation of
STAT3 signaling.322 MiR-30 targets SOCS3 in glioma stem cells.337
Mir-93 downregulates the expression of JAK1 and STAT3 to induce
the differentiation of breast CSCs. Mir-218 negatively regulates the
IL-6 receptor and JAK3 gene expression in lung CSCs.338 In
addition, some endogenous or exogenous genes inhibit JAK/STAT
signaling in CSCs. Von Hippel–Lindau suppresses the tumorigeni-
city and self-renewal ability of glioma stem cells by inhibiting
JAK2/STAT3.323 Although there are few studies on JAK in CSCs,
there is a role for JAK/STAT signaling in the survival, self-renewal,
and metastasis of CSCs.
TGF/SMAD signaling pathway in CSCs. The TGF-β signaling
pathway is involved in many cellular processes associated with
organism and embryo development, including cell proliferation,
differentiation, apoptosis, and homeostasis. Although the TGF-β
signaling pathway regulates a wide range of cellular processes, its
structure is relatively simple. TGF-β superfamily ligands bind to a
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type II receptor, which recruits a type I receptor and phosphor-
ylates it. This type I receptor phosphorylates receptor-regulated
Smads (R-Smads), which bind to common pathway Smad (co-
Smad). The R-Smad/co-Smad complex acts as a transcription
factor and accumulates in the nucleus to regulate the expression
of target genes. TGF-β superfamily ligands include BMPs, growth
and differentiation factors (GDFs), anti-Mullerian hormone (AMH),
activin Nodal, and TGF-β.339 These ligands can be divided into two
groups, TGF-β/activin and BMP/GDF. The TGF-β/activin group
includes TGF-β, activin, and Nodal, and the BMP/GDF group
includes BMP, GDF, and AMH ligands.340 Based on Smad structure
and functions, Smad proteins can be divided into three
subfamilies: receptor-activated or pathway-restricted Smad (R-
Smads), Co-Smad, and inhibitory Smad (I-Smads), which includes
at least nine Smad proteins.341,342 R-Smads are activated by type I
receptors and form transient complexes with these receptors.
There are two types of Smad complexes: AR-Smads are activated
by activin TGF-β, including Smad2 and Smad3, and BR-Smads are
activated by BMP, including Smad1, Smad5, Smad8, and Smad9.
Co-Smad, including Smad4, is a common medium in various TGF-β
signal transduction processes. I-Smads, including Smad6 and
Smad7, bind to activated type I receptors and inhibit or regulate
signal transduction of the TGF-β family.343
Many studies have shown that activation of TGF/Smad signaling
also occurs in human cancers. Dkk-3, a secreted protein, inhibits
TGF-β-induced expression of matrix metallopeptidase 9 (MMP9)
and MMP13 to prevent migration and invasion of prostate
cancer.344 Cancer upregulated gene 2 promotes cellular transfor-
mation and stemness, which is mediated by nuclear NPM1 protein
and TGF-β signaling in lung cancer.345 TGF/Smad also plays an
important role in the cell proliferation of CSCs. Cyclin D1 interacts
with and activates Smad2/3 and Smad4, promoting cyclin D1-
Smad2/3-Smad4 signaling to regulate self-renewal of liver CSCs.346
CD51 binds to TGF-β receptors to upregulate TGF-β/Smad
signaling in colorectal CSCs.341 Upregulation of TGF-β1 induces
the expression of smad4, p-Smad2/3, and CD133 in liver CSCs.347
TGF-β1 also upregulates the expression of PFKFB3 through
activation of the p38 MAPK and PI3K/Akt signaling pathways to
regulate glycolysis in glioma stem cells.348 Furthermore, silencing
ShcA expression also induces activation of STAT4 in breast
CSCs.349 Moreover, miR-148a inhibits the TGF-β/Smad2 signaling
pathway in HCC stem cells.350 Smad7, a newly discovered target
gene of miR-106b, is an inhibitor of TGF-β/Smad signaling, which
inhibits sphere formation of gastric cancer stem-like cells.351
Although there are few studies on the TGF/Smad signaling
pathway in CSCs, this pathway still plays a very important role.
PI3K/AKT/mTOR signaling pathway in CSCs. Phosphatidylinositol-
3-kinase (PI3K) is an intracellular phosphatidylinositol kinase.352 It
consists of the regulatory subunit p85 and catalytic subunit p110,
which have serine/threonine (Ser/Thr) kinase and phosphatidyli-
nositol kinase activities.353 AKT is a serine/threonine kinase that is
expressed as three isoforms: AKT1, AKT2, and AKT3.354 AKT
proteins are crucial effectors of PI3K and are directly activated in
response to PI3K. One of the key downstream target genes of AKT
is the mammalian target of rapamycin (mTOR) complex, which is a
conserved serine/threonine kinase. It forms two distinct multi-
protein complexes: mTORC1 and mTORC2.355 mTORC1 consists of
mTOR, raptor, mLST8, and two negative regulators, PRAS40 and
DEPTOR.356,357 mTORC2 phosphorylates AKT at serine residue 473,
which leads to full AKT activation.358
Studies show that mutations in PTEN lead to the inhibition of
PI3K/mTOR signaling in glioblastoma multiforme. However,
deletion of PTEN in neural stem cells leads to a neoplastic
phenotype that includes cell growth promotion, resistance to cell
apoptosis, and increased migratory and invasive properties
in vivo.359 Inactivation of PTEN and activation of protein kinase
B have been found in other solid tumors, such as
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myeloproliferative neoplasia and leukemia.360 Therefore, the PI3K/
mTOR signaling pathway is vital for cell proliferation and survival.
Abnormal activation of PI3K/mTOR signaling is found in some
cancers, such as non-small-cell lung cancer,361 breast cancer,362
prostate cancer,363 Burkitt lymphoma,364 esophageal adenocarci-
noma,365 and colorectal cancer.366
Although PI3K/AKT/mTOR has been extensively studied in
cancers, there are few studies in CSCs.358 PI3K/Akt/mTOR signaling
is involved in ovarian cancer cell proliferation and the
epithelial–mesenchymal transition.367 This signaling activation
also enhances the migration and invasion of prostate and
pancreatic CSCs.368,369 Downregulation of PTEN induces PI3K
activation to promote survival, maintenance of stemness, and
tumorigenicity of CD133+/CD44+ prostate cancer stem-like cell
populations.370 PI3K activation promotes cell proliferation, migra-
tion, and invasion in ALDH+CD44high head and neck squamous
CSCs.371 Activation of mTOR promotes the survival and prolifera-
tion of breast CSCs and nasopharyngeal carcinoma stem
cells.328,372 mTORC1 activation also increases aldehyde dehydro-
genase 1 (ALDH1) activity in colorectal CSCs.373 Activation of
mTORC2 upregulates the expression of the hepatic CSC marker
EpCAM (epithelial cellular adhesion molecule) and tumorigenicity
in hepatocellular CSCs.374 Nucleotide-binding domain and leucine-
rich repeats (NLRs) belong to a large family of cytoplasmic sensors.
NLRC3 (also known as CLR16.2 or NOD3) is associated with PI3Ks
and blocks activation of PI3K-dependent kinase AKT in colorectal
CSCs.375
In addition, some studies have shown that the mTOR signaling
pathway is closely related to the metabolism of CSCs. For example,
low folate (LF) stress reprograms metabolic signals through the
activated mTOR signaling pathway, promoting the metastasis and
tumorigenicity of lung cancer stem-like cells.376 However, matcha
green tea (MGT), an inhibitor of mTOR, inhibits the proliferation of
breast CSCs by targeting mitochondrial metabolism, glycolysis,
and multiple cell signaling pathways.377 A link between the PI3K/
Akt/mTOR pathway and CSCs is clearly evident.
PPAR signaling pathways in CSCs. Peroxisome proliferator-
activated receptors (PPARs) are ligand-activated nuclear transcrip-
tion factors that were first cloned from mouse liver by Isseman
and Green.378 PPARs are also members of the ligand-activated
transcription factor superfamily of nuclear hormone receptors that
are associated with retinoic acid, steroids and thyroid hormone
receptors. PPARs act as fat sensors to regulate the transcription of
lipid metabolic enzymes.379 At present, three subtypes, PPARα,
PPARβ, and PPARγ (encoded by the PPARA, PPARD, and PPARG
genes, respectively), have been found.380 PPARα is highly
expressed in hepatocytes, cardiac myocytes, intestinal cells, and
renal proximal convoluted tubule cells. PPARγ is abundantly
expressed in adipose tissue, vascular parietal cells (such as
monocytes/macrophages, ECs, and smooth muscle cells), and
myocardial cells.381 PPARβ is expressed in almost all tissues of the
body, and its expression level is higher than that of PPARα or
PPARγ.382 In recent years, studies have found that PPARs are
closely related to energy (lipid and sugar) metabolism, cell
differentiation, proliferation, apoptosis, and inflammatory reac-
tions.383 PPARs can exert positive or negative effects to regulate
target gene expression by binding to a specific peroxisome
located at each gene regulatory site and a proliferative response
element.378 Their natural ligands are unsaturated fatty acids,
eicosane acids, oxidized low-density lipoprotein, very low-density
lipoprotein, and linoleic acid derivatives.384
To date, there have been many reports about the role of PPARs
in cancer cells, including prostate cancer, breast cancer, glioblas-
toma, neuroblastoma, pancreatic cancer, hepatic cancer, leukemia,
and bladder cancer and thyroid tumors.385 However, the function
of PPARs in CSCs is not well understood, except for some reports
on PPARγ. As a tumor suppressor, PPARγ binds and activates a
canonical response element in the miR-15a gene in breast CSCs to
reduce the CD49high/CD24+ mesenchymal stem cell (MSC)
population and inhibit angiogenesis.386 PPARγ activation also
prevents cell spheroid formation and stem cell-like properties in
bladder CSCs and induces adipocyte differentiation and β-catenin
degradation in adipose tissues.387 Furthermore, expression of
PPARγ restrains YAP transcriptional activity to induce differentia-
tion in osteosarcoma stem cells388 and melanoma cells.389 The
PPARγ/NF-κB pathway promotes M2 polarization of macrophages
to prevent cell death in ovarian CSCs4.390 PPARγ activation
promotes expression of its target gene PTEN to inhibit PI3K/Akt/
mTOR signaling, which stunts self-renewal, tumorigenicity, and
metastasis in cervical CSCs, glioblastoma stem cells, and liver
CSCs.391,392 However, combined expression of Dnmt3a and
Dnmt3b inhibits PPARγ expression by direct methylation of its
promoter in squamous carcinomas.393 PPARs are also closely
related to the metabolism of CSCs. PPARα and PPARβ/δ regulate
metabolic reprogramming in glioblastoma stem cells, lung CSCs,
and mouse mammary gland cancer.394 The transcription coacti-
vator peroxisome proliferator-activated receptor gamma coacti-
vator 1α (PPARGC1A, also known as PGC-1α) promotes CSC
proliferation and invasion by enhancing oxidative phosphoryla-
tion, mitochondrial biogenesis, and the oxygen consumption rate
of breast CSCs.395 In addition, the AMPK signaling pathway
(adenosine 5′-monophosphate (AMP)-activated protein kinase) is
an AMP-dependent protein kinase that is a key molecule in the
regulation of bioenergy metabolism and is the core of the study of
diabetes and other metabolic-related diseases. AMPK is expressed
in various CSCs related to metabolism. Some studies have shown
that AMPK is necessary for prostate CSCs to maintain glucose
balance.396 Metformin, an antidiabetic drug that fights cancer,
targets AMPK signaling to inhibit cell proliferation and metabolism
in colorectal CSCs397 and HCC stem cells.398 Therefore, metformin
may be a potential therapeutic regent by regulating the energy
metabolism of CSCs. These studies suggest that PPARs play an
important role in the growth of CSCs.
Interactions between signaling pathways in CSCs. As mentioned
previously, these complex signal transduction pathways are not
linear. In some cases, crosstalk between and among various
pathways occurs to regulate CSCs.399 Wnt/β-catenin and NF-κB
signaling work together to promote cell survival and proliferation of
CSCs. TNFRSF19, a member of the TNF receptor superfamily, is
regulated in a β-catenin-dependent manner, but its receptor
molecules activate NF-κB signaling to regulate the development of
colorectal cancer.400 Knockdown of CD146 results in inhibition of NF-
κB/p65-initiated GSK3β expression, which promotes nuclear translo-
cation and activation of β-catenin.401 In addition, there is negative
regulation between Wnt/β-catenin and NF-κB signaling. Studies
have revealed a negative effect of β-catenin on NF-κB activity in
liver, breast, and colon cancer cells.402,403 Leucine zipper tumor
suppressor 2 (LZTS2) is a putative tumor suppressor, and NF-κB
activation inhibits β-catenin/TCF activity through upregulation of
LZTS2 in liver, colon, and breast cancer cells.404–406 Wnt/β-catenin
and Hh signaling have important functions in embryogenesis, stem
cell maintenance, and tumorigenesis. Wnt/β-catenin signaling
induces the expression of CRD-BP, an RNA-binding protein, which
results in the binding and stabilization of Gli1 mRNA, leading to an
increase in Gli1 expression and transcriptional activity, which
promotes the survival and proliferation of colorectal CSCs.407
However, a report showed that noncanonical Hh signaling is a
positive regulator of Wnt signaling in colon CSCs.408
In addition, crosstalk between pathways promotes cell growth
and metastasis through maintenance of the CSC population.
Downregulation of Notch1 and IKKα enhances NF-κB activation to
promote the CD133+ cell population in melanoma CSCs.409 IL-6/JAK/
STAT3 and TGF-β/Smad signaling induce the proliferation and
metastasis of lung CSCs.410 IL-17E binding to IL-17RB activates the
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Fig. 4 The microenvironment of cancer stem cells. Proliferation, self-renewal, differentiation, metastasis, and tumorigenesis of CSCs in the CSC
microenvironment. The CSC microenvironment is mainly composed of vascular niches, hypoxia, tumor-associated macrophages, cancer-
associated fibroblasts, cancer-associated mesenchymal stem cells, and extracellular matrix. These cells in response to hypoxic stress and
matrix induce growth factors and cytokines (such as IL-6 and VEGF) to regulate the growth of CSCs via Wnt, Notch, and other signaling
pathways
NF-κB and JAK/STAT3 pathways to promote proliferation and sustain
self-renewal of CSCs in HCC.411 TGF-β1 silencing decreases the
expression of Smad2/3, β-catenin, and cleaved-Notch1 to inhibit the
activation of Wnt and Notch signaling in liver CSCs.346 Activation of
TGF-β1 induces lncRNA NKILA expression to block NF-κB signaling,
which inhibits metastasis of breast CSCs.412 TGF-β also directly
regulates the expression of Wnt5a in breast CSCs to limit the stem
cell population.413 Furthermore, Notch, IKK/NF-κB, and other path-
ways together regulate the proliferation and metastasis of CD133+
cutaneous SCC stem cells.409 PI3K/mTOR signaling upregulates the
expression of STAT3 to promote the survival and proliferation of
breast CSCs.328 Inhibition of TORC1/2 increases FGF1 and Notch1
expression. The PI3K/AKT/mTOR and Sonic Hh pathways cooperate
to inhibit the growth of pancreatic CSCs.414 Increasing evidence
shows that crosstalk regulates the survival, self-renewal, and
metastasis of CSCs.
The microenvironment of CSCs
CSCs interact with the microenvironment through adhesion
molecules and paracrine factors. The microenvironment provides
a suitable space for the self-renewal and differentiation of CSCs,
protects CSCs from genotoxicity, and increases their chemical and
radiological tolerance. The TME mainly consists of the tumor
stroma, adjacent tissue cells, microvessels, immune cells, and
immune molecules.415 CSCs not only adapt to changes in the TME
but also affect the TME. Concurrently, the microenvironment also
promotes the self-renewal of CSCs, induces angiogenesis, recruits
immune and stromal cells, and promotes tumor invasion and
metastasis (Fig. 4).
Vascular niche microenvironments and CSCs. The normal vascu-
lature is composed of ECs, basement membranes, and parietal cells.
ECs are the basis for the formation of the inner surface of blood
vessels.416 Studies reported that glioblastoma stem cells are located
around the blood vessels, and the concept of the cancer
microvascular environment was first proposed. Calabrese et al.417
demonstrated that direct contact between ECs and CSCs occurs in
brain tumors. CSCs are also found near ECs in other cancers, such as
papilloma and colorectal cancer.418,419 A study also showed that
CD133+/CD144− glioma stem cell-like cells differentiate into cancer
cells and endothelial progenitor cells and finally into mature ECs.420
CSCs differentiate into cancer vascular stem cells/progenitor cells and
are directly involved in angiogenesis or form vasculogenic mimicry
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that is directly involved in the microcirculation of tumors.421,422 ECs
also promote CSC-like transformation and cell growth through Shh
activation of Hh signaling.423 Moreover, secreted microvesicles of
CSCs promote the proliferation of human umbilical vein ECs and
form a tube-like structure in vitro and in vivo in mice.424–426 This CSC
plasticity has also been demonstrated in other tumors, including
neuroblastoma, renal, breast, and ovarian cancer.427–430
The vascular microenvironment maintains the initial undifferen-
tiated dormancy of stem cells, supports self-renewal, invasion and
metastasis of CSCs, and protects CSCs from any injury.431 The role of
the EC signaling system has been proven in maintaining the survival
and self-renewal of head and neck SC stem cells.432 Pasquier and
colleagues433 showed that treatment with EC microparticles in breast
and ovarian cancer models increased the number of CSCs and
promoted sphere formation of CSCs. The interaction between CSCs
and blood vessels promotes the self-renewal of CSCs through the
VEGF-Nrp1 loop.418 CSCs promote cancer angiogenesis by inducing
secretion of the cytokines VEGF and hepatocyte growth factor (HGF)
from ECs.434 VEGF receptor 2 plays a key role in vasculogenic mimicry
formation, neovascularization, and tumor initiation of glioma stem-
like cells.435 As a result, the secretion of VEGF in stem cell-like glioma
cells is higher than that in normal cancer cells424 and regulates the
proliferation of glioma stem cells through the mTOR signaling
pathway.436 Subsequent studies have further shown that multiple
signals, such as integrin, Notch, and growth factor receptors, are
linked to each other on the cell surface to maintain the stemness of
CSCs.437,438
The hypoxia microenvironment and CSCs. Hypoxia is a key
component for CSC formation and maintenance.439 The hypoxic
microenvironment maintains the undifferentiated state of cancer
cells, enhances their cloning rate, and induces the expression of
CD133 as a specific biomarker of CSCs.440 HIFs are important
transcription factors that regulate cellular hypoxia responsive-
ness441 and inhibit cell apoptosis.442 As a heterodimer, HIF is
composed of HIFα and HIFβ.443 HIF-1α regulates the proliferation
and fate of CSCs in medulloblastoma and glioblastoma multi-
forme444 and activates the NF-κB pathway to promote CSC
survival and tumorigenesis.445 HIF-2α maintains the survival and
phenotype of CSCs.446 HIFα also regulates the expression of the
target genes GLUT1, GLUT3, LDHA, and PDK1. Thus, CSCs can
adapt to a new method of cell energy metabolism and avoid
apoptosis caused by hypoxia.447
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HIFs also regulate the stemness of CSCs. Previous studies have
shown that CSCs need to activate HIF-1α and HIF-2α to maintain
their self-sustainability under hypoxic conditions448 and obtain
pluripotency by upregulating the Sox2 and Oct4 genes.440 More
importantly, activation of C-MYC by HIF-2α is necessary to ensure
undifferentiated CSCs.449 The Wnt and Notch signaling pathways
regulated by hypoxia and can induce the EMT, which promotes
the stemness of CSCs and increases the invasiveness and
resistance to radiotherapy and chemotherapy.450 HIF-1α binds
the Notch ICD and enhances its transcriptional activity. In the
hypoxic microenvironment of glioma, both HIF-1α and HIF-2α
require the Notch signaling pathway to ensure the self-renewal
and undifferentiated status of CSCs.451
Tumor-associated macrophages and CSCs. Macrophages are an
important component of the innate immune response and are a
group of cells with plasticity and heterogeneity.452 Infiltrating and
inflammatory macrophages originate from the precursors of bone
marrow mononuclear cells.453 These precursor cells infiltrate
various tissues from blood vessels and differentiate into different
subtypes in different microenvironments. There are two subtypes
of macrophages: the M1 and M2 phenotypes. The M1 phenotype
has anti-inflammatory and anti-tumor effects and secretes
proinflammatory factors such as interleukin-1 (IL-1), IL-12, IL-23,
TNF-α, chemokine (C-X-C motif) ligand 5 (CXCL5), CXCL9, and
CXCL10. M2 macrophages are generally considered to be the
phenotype of tumor-associated macrophages (TAMs),454–456 have
immunosuppressive and angiogenesis-promoting effects, and are
considered to be a tumor-promoting cell type.456,457 M2 macro-
phages secrete CCL17 (C-C chemokine ligand 17), CCL22, and
CCL24 and have low expression of IL-12 and high expression of IL-
10. Cytokines secreted by macrophages affect the proliferation,
tumorigenic transformation, or apoptosis of CSCs through various
signaling pathways.458
TAMs are closely related to CSCs or stem cell transformation.
Renal epithelial cells cocultured with macrophages induce the
EMT to transform renal cancer cells into CSCs expressing CD117,
Nanog, and CD133.459 Another study also showed that mucin-1
secreted by M2 macrophages induces the transdifferentiation of
non-small-cell lung cancer cells into CSCs that express CD133 and
Sox2.460 Jinushi and colleagues461 also reported that TAMs secrete
MFG-E8, which maintains the self-renewal ability of colon and
breast CSCs, and knockout of MFG-E8 significantly inhibits the
tumorigenic ability in SCID mice.461 TAMs are closely related to
glioma stem cell growth.462 TAMs are mainly distributed near
CD133+ glioma stem cells and accumulate in pericapillary and
hypoxic areas.463 Glioma stem cells recruit and maintain macro-
phages by secreting a potent chemokine membrane protein.464
The ablation of TAMs inhibits the tumorigenesis of glioma stem
cells.465 Recent studies have shown that the interaction between
the TME and CSCs is regulated by a variety of signaling
pathways.466 Macrophages enhance the invasion of glioma
stem-like cells through the TGF-β1 signaling pathway.467 TAMs
activate the STAT3/Sox2 signaling pathway in mouse breast CSCs
by secreting EGF, which promotes the self-renewal ability of
CSCs.468 IL-8 secreted by TAMs also induces the EMT in
hepatocellular cancer cells by activating the JAK2/STAT3/Snail
pathway.469
Cancer-associated fibroblasts and CSCs. CAFs are one of the most
important components of the TME and are critical in tumor
development and metastasis.470 The origin of these cells in the
stroma is not entirely clear. Current studies hypothesize that there
are five possible sources: (1) transference of fibroblasts in the host
stroma;471 (2) EMT;472 (3) transdifferentiation of perivascular
cells;473 (4) EMT;474 and (5) differentiation of MSCs derived from
bone marrow.475 In addition, CAFs are also derived from other cell
types, such as smooth muscle cells, pericytes, adipocytes, and
immune cells.476 It is not clear whether there are differences in the
functions of CAFs from different sources. CAFs affect cancer cell
growth through cell–cell interactions and the secretion of various
invasive molecules, such as cytokines, chemokines, and inflam-
matory mediators.477–479
CAFs in the TME play an indispensable role in the generation
and maintenance of CSCs.480 CAFs transform cancer cells into
CSCs.481 Studies have shown that CAFs promote the EMT and
enhance the expression of prostate CSC markers482 by secreting
IL-6 and IL-1β in breast cancer.483,484 CAFs also secrete TGF-β and
activate related pathways to increase ZEB1 transcription, which
stimulate lung cancer cells to undergo EMT and CSC transforma-
tion.485 CAFs secrete matrix metalloproteinases, which induce the
EMT and promote the growth of stem cell-specific components in
tumors.482 Paracrine interaction between CAFs and CSCs is critical
for maintaining the CSC niche of lung CSCs.486 Fibroblast-derived
CCL-2 regulates CSCs through gap activation, thus promoting the
progression of tumors.487 CAFs and adipocytes also secrete leptin,
which increases the globulation rate of breast CSCs in vitro.488
CAFs also regulate the proliferation of CSCs by other signaling
pathways. For example, CAFs increase the secretion of CCL-2 to
activate the Notch1/STAT3 pathway, which increases the expres-
sion of stem cell markers and upregulates the globulation rate in
breast cancer.489 CAFs regulate TIC plasticity in HCC through c-
Met/FRA1/HEY1 signaling.490 CAFs secrete high levels of IL-6 to
activate Notch signaling through STAT3 Tyr705 phosphorylation,
thus promoting the stem cell-like characteristics of HCC cells.491
Similar studies have shown that CAF-derived exons enhance colon
stem cell resistance to 5-fluorouracil by activating the Wnt
signaling pathway.492
Cancer-associated MSCs and CSCs. MSCs have high self-renewal
ability and multidirectional differentiation potential.493 MSCs also
specifically migrate to the injured site and tumor tissue and are
easy to isolate and expand in vitro.494,495 MSCs are considered to
be an ideal vector for gene therapy because of their characteristics
of homing to and secreting cytokines in tumors.496 However,
these tumorigenic characteristics of MSCs still need to be studied.
MSCs not only promote tumor development497,498 but also inhibit
cancer cell growth.499 Bone marrow MSCs promote tumor growth
by promoting angiogenesis, metastasis, and the survival of
CSCs.500 MSCs in the TME are conducive to the proliferation,
carcinogenesis, and metastasis of breast CSCs through ionic
purinergic signal transduction.501 MSCs can differentiate into
CAFs, and CAFs further regulate CSCs and promote the occurrence
and metastasis of cancers.502 The possible mechanism is related to
the spontaneous fusion between cancer cells and MSCs.503 The
fusion of MSCs with breast cancer, ovarian cancer, gastric cancer,
and lung cancer cells in vitro and in vivo has been con-
firmed.504,505 MSCs regulate the TME by secreting IL-6 to maintain
the undifferentiated state of osteosarcoma cells.506,507 IL-1
stimulates the secretion of PGE2 via autocrine signaling, which
ultimately activates β-catenin signaling in cancer cells in a
paracrine manner and transforms cancer cells into CSCs.508 In
the ECM, bone mesenchymal stem cells activate the NF-κB
pathway and induce a CSC phenotype by secreting a variety of
cytokines and chemokines, such as CXCL12, CXCL7, and IL-6/IL-
8.509 The interaction between MDSCs and CSCs via IL-6/STAT3 and
Notch signaling is critical to the progression of breast cancer.510
Extracellular matrix and CSCs. The ECM is an insoluble structural
component of the matrix in mesenchymal and epithelial vessels.
The ECM includes collagen, elastin, aminoglycan, proteoglycan,
and noncollagen glycoprotein.511,512 At present, increasing
evidence shows that the ECM is an integral part of stem cell
niches that regulates the balance of stem cells in three different
biological states: static, self-renewal, and differentiation.513 Experi-
ments in vitro and in vivo have shown that ECM receptors can be
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used to aggregate CSCs514 and induce drug resistance.513,515
Fibronectin, vimentin, collagen, and proteoglycan in the ECM bind
to cytokines such as FGF, HGF, VGF, BMP, and TGF-β in the TME
and regulate their activities.516 In HCC, an increased matrix
promotes cell proliferation and chemotherapeutic resistance and
increases the expression of CSC-related markers, including CD44,
CD133, c-kit, cxcr4, Oct4, and Nanog. Hyaluronic acid in the ECM is
a ligand for the CD44 receptor and can regulate the acquisition
and maintenance of CSC stemness during mutual contact.517 The
ECM also binds the Wnt ligand Wnt5b via molecular MMP3 and
leads to the expansion and proliferation of mammary epithelial
stem cells.518 In addition, tenascin C in the ECM maintains the
stability of breast CSCs by increasing the activity of the Wnt and
Notch signaling pathways.519
Exosomes in the TME and CSCs. Exosomes are nanovesicles
secreted by various types of living cells (30–100 nm in
diameter)520 and are widely distributed in peripheral blood, saliva,
urine, ascites, pleural effusion, breast milk, and other body
fluids.521 Exosomes contain a large number of functional proteins,
RNA, microRNAs, DNA fragments, and other bioactive sub-
stances.522–525 These bioactive substances mediate material
transport and information exchange between cells, thus affecting
the physiological function of cells.526,527 The exosomes secreted
by cancer cells promote angiogenesis,528 induce differentiation of
tumor-related fibroblasts,529 participate in immune regulation of
the TME,530 and regulate the microenvironment before metas-
tasis.531 Clinical analysis has revealed that exosomes are released
at higher levels in cancer cells.532
Recent studies have shown that endocytosis of lipid rafts in MSCs
is associated with increased secretion of exosomes.533 Exosome
signaling mediates the interaction of CSCs and normal stem cells,
thereby regulating oncogenesis and tumor development.534 Exo-
somes also regulate CSC growth by targeting specific signaling
pathways, such as Wnt, Notch, Hippo, Hh, and NF-κB.535–537
Extracellular vesicles released by glioblastoma stem cells promote
neurosphere formation, endothelial tube formation, and the
invasion of glioblastoma.538 CSCs promote cell proliferation and
self-renewal through crosstalk between exosome signal transduction
and the surrounding microenvironment.539 The exosomes released
from CSCs affect signal transduction in nearby breast cancer cells540
and increase the stemness of breast cancer cells.540 Similarly,
fibroblast-derived exosomes contribute to chemoresistance by
promoting colorectal CSC growth.491 Exosomes in the TME promote
the transformation of non-CSCs into CSCs. CAF-derived exosomes
significantly increase the ability to form mammary globules and
promote the stemness of breast cancer cells.541 Similarly, CAF-
derived exosomes also promote sphere formation of colorectal
cancer cells by activating Wnt signaling and ultimately increase the
percentage of CSCs.491 Exosomes from glioma-associated MSCs
increase the tumorigenicity of glioma stem-like cells by transferring
miR-1587.542 In addition, exosomes regenerate stem cell phenotypes
by mediating the EMT or regulating stem cell-related signaling
pathways, such as the Wnt pathway, Notch pathway, Hh pathway
and other pathways, which convert cells into CSCs.543 Exosomes
have many advantages, such as low immunogenicity, biocompat-
ibility, easy production, cytotoxicity, easy storage, high drug loading
capacity, and long life and have become ideal drug carriers for
cancer therapy.544–548
THERAPEUTIC TARGETING OF CSCS
Agents targeting CSC-associated surface biomarkers in clinical
trials
Monoclonal antibodies (mAbs) that target CSC-specific surface
biomarkers have become an emerging technology for cancer
therapy. Rituximab, a CD20 mAb, is an active agent for the
treatment of follicular lymphoma and mantle-cell lymphoma, but
Signal Transduction and Targeted Therapy (2020)5:8
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Yang et al.
15
some serious adverse reactions still occur.549 Subsequently, to
improve the availability and affordability of radioimmunotherapy
for refractory or recurrent non-Hodgkin’s lymphoma (NHL), a
phase II clinical trial for a radioiodine replacement of rituximab
was carried out, which showed a response rate of 71% and a
complete remission rate of 54% in 35 patients, with only two cases
of grade IV hematologic toxicity observed.550 Encouragingly,
alemtuzumab, a humanized CD52 antibody, has been approved
for the treatment of chronic lymphocytic leukemia (CLL) in
patients who failed to respond to alkylating agents and purine.
Furthermore, the combination of the CD20 and CD52 antibodies in
the treatment of refractory CLL was safe, nontoxic, feasible, and
positive.551 Another antibody drug, relabeled bivatuzumab, is an
anti-CD44v6 mAb,71 which was found to be safe when it was used
for the treatment of head and neck SCC.552 These results have
been obtained in subsequent clinical research553 and safety/
efficacy studies.554 Unfortunately, in a stage I dose escalation
study with the CD44v6 antibody, one patient with head and neck
SCC of the esophagus suffered deadly skin toxicity.555
Several CD123 antibodies have been developed, XmAb14045
and MGD006, and were designed with biospecific effects
against CD3 and CD123. Talacotuzumab is also effective against
CD16 and CD123. CSL360, another CD123 antibody, was used to
treat relapsed, refractory, or high-risk acute myeloid leukemia
(AML) and displayed no anti-leukemic activity in most cases.556
SL-401, another CD123 antibody, was used to treat blastic
plasmacytoid dendritic cell neoplasm patients. The results
showed major positive responses in seven out of nine patients,
including five complete responses and two partial responses.557
An ongoing phase II study of SL-401 in 29 patients with blastic
plasmacytoid dendritic cell neoplasms demonstrated robust
single-agent activity with an 86% overall response rate.558 The
latest antibodies against CSC surface markers, such as
XmAb14045 (NCT02730312), flotetuzumab (NCT02152956),
and talacotuzumab (NCT02472145), are also in clinical study.
Furthermore, several other markers that can distinguish LSCs
from other cells are under clinical development, such as IL-1
receptor accessory protein, CD27/70, CD33, CD38, CD138, CD93,
CD99, C-type lectin-like molecule-1, and T cell immunoglobulin
mucin-3.
EpCAM, a common CSC biomarker, has also received attention
in clinical trials.559 Adecatumumab, an EpCAM antibody, was used
in patients with hormone-resistant prostate cancer, and the results
showed that the EpCAM-specific antibody has great clinical
potential.560 Catumaxomab, a multifunctional mAb against
EpCAM, binds and recognizes EpCAM and the T cell antigen
CD3 (anti-EpCAM × anti-CD3).561 Intraperitoneal injection of catu-
maxomab to treat EpCAM-positive ovarian cancer and malignant
ascites has shown high efficacy in killing cancer cells and reducing
the formation of ascites.562 Catumaxomab has been used in non-
small-cell lung cancer and also had a good survival rate.561
However, other types of EpCAM antibodies, such as edrecolo-
mab563 and adecatumumab,564 have minimal efficacy in colorectal
and breast cancers. Combining EpCAM antibodies with chimeric
antigen receptor T cell (CAR-T) technology has also been used in
various types of cancers in phase I trials, such as NCT02915445,
NCT03563326, NCT02729493, and NCT02725125. With a deeper
understanding of CSC surface biomarkers, there has been
significant progress in developing antibodies targeting CSCs
(Table 2). However, CSC surface phenotypes can vary in different
patients or different cancers, and different CSC populations with
different phenotypes might coexist. CSCs also diverge or evolve
into different cancer cells, acquiring distinct phenotypes upon
relapse. Therefore, the strategies used in clinical trials should be
determined according to the phenotypes of the different cancers.
At the same time, combining different surface antibodies with
relevant chemotherapy drugs can achieve an ideal therapeutic
effect.
 Targeting cancer stem cell pathways for cancer therapy
Yang et al.
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Table 2. Agents targeting CSC-associated surface markers in ongoing clinical trials

Drug name Antibody target Condition Sample size Highest status NCT number Current status

Surface antigens
Catumaxomabr (emovab) EpCAM/CD3 Ovarian cancer II 44 NCT00189345 Completed
Tagraxofusp CD123 Acute myeloid leukemia I 36 NCT03113643 Recruiting
SL-401
KHK2823 I 39 NCT02181699 Terminated
Talacotuzumab III 326 NCT02472145 Completed, has results
SGN-CD123A I 17 NCT02848248 Terminated
IMGN632 II 155 NCT03386513 Recruiting
XmAb14045 CD123/CD4 II 105 NCT02730312 Recruiting
MGD006 CD123/CD3 II 179 NCT02152956 Recruiting
JNJ-63709178 III 326 NCT02472145 Completed, has results
CSL362 CD124 I 30 NCT01632852 Completed
TTI-621 CD47 Solid tumor I 260 NCT02663518 Recruiting
Hu5F9-G4 Solid tumor I 88 NCT02216409 Completed
IBI188 Advanced malignancies I 42 NCT03763149 Recruiting
CC-90002 Hematologic neoplasms I 28 NCT02641002 Terminated
AO-176 Solid tumor I 90 NCT03834948 Recruiting
SRF231 Solid tumor I 148 NCT03512340 Recruiting
Bivatuzumab mertansine Metastatic breast cancer I 24 NCT02254005 Completed
Vadastuximab talirine (SGN-CD33A) CD33 Acute myelogenous leukemia I 195 NCT01902329 Completed
IMGN779 I 62 NCT02674763 Completed
Mylotarg (gemtuzumab ozogamicin) ECG IV 56 NCT03727750 Recruiting
RO5429083 CD44 Malignant solid tumors I 65 NCT01358903 Completed
SPL-108 Ovarian cancer I 18 NCT03078400 Recruiting
Salazosulfapyridine CD44V4 Non-small-cell lung cancer I UMIN000017854
AMC303 CD44V6 Solid tumor I 55 NCT03009214 Recruiting
Immune checkpoints
Ipilimumab CTLA-4 Non-small-cell lung cancer II 24 NCT01820754 Completed, has results
Nivolumab PD-1 Glioblastoma multiforme II 29 NCT02550249 Completed
Pembrolizumab II 80 NCT02337491 Completed, has results
Cemiplimab II 30 NCT04006119 Recruiting
Idarubicin Acute myeloid leukemia II 51 NCT01035502 Completed
Sym021 Solid tumor lymphomas I 102 NCT03311412 Recruiting
Durvalumab Solid tumors II 124 NCT02403271 Completed, has results
Atezolizumab PD-L1 Non-small-cell lung cancer III 1225 NCT02008227 Completed, has results
Avelumab Recurrent glioblastoma II 52 NCT03291314 Completed
Sym023 Tim3 Solid tumor I 48 NCT03489343 Recruiting
ARGX-110 CD70 Acute myeloid leukemia II 36 NCT03030612 Active, not recruiting
Varlilumab (CDX-1127) Solid tumors II 175 NCT02335918 Completed
Sym022 LAG3 Solid tumor I 30 NCT03489369 Recruiting
MGD013 CD70/LAG3 Solid tumors I 255 NCT03219268 Recruiting

Agents targeting CSC-associated signaling pathways in clinical
trials
The signaling pathways that regulate the maintenance and
survival of CSCs have become targets for cancer treatment. At
present, the main signaling pathways are the Wnt, Notch, and Hh
signaling pathways, as well as the TGF-β, JAK-STAT, PI3K, and NF-
κB signaling pathways. These pathways often interact with each
other during tumor development and in CSCs. Considerable
progress has been made in early clinical trials for Notch and Hh
pathway inhibitors, while targeting the Wnt pathway has proven
to be difficult.10
The Notch signaling pathway plays an important role in the
maintenance of CSCs565,566 and can induce CSC differentiation.
Abnormal activity of the Notch signaling pathway has been
observed in many cancers, such as leukemia,567 glioblas-
toma,568,569 breast cancer,570 lung cancer,571 ovarian cancer,572
pancreatic cancer,573 and colon cancer.574 At present, there are
three major clinical methods used to inhibit Notch signaling,
secretase inhibition (γ-secretase inhibitor (GSI)), Notch receptor
or ligand antibodies, and combination therapy with other
pathways. For example, GSIs have been tested in clinical trials.
Among them, MK-0752 (NCT00100152) was the first GSI used to
treat T cell acute lymphoblastic leukemia in children in a phase I
trial. However, the study was terminated because of poor
results.575 MK-0752 also had no clinical activity in extracranial
solid tumors in subsequent phase II trials. Only one complete
response with interdegenerative astrocytoma and SD extension
out of 10 patients with different types of glioma was
observed.576 MK-0752 is well tolerated and shows targeted
inhibition in recurrent pediatric central nervous system
tumors.577 In addition, combining MK-0752 with cisplatin
treatment for ovarian cancer,578,579 docetaxel treatment for
locally advanced or metastatic breast cancer,569 and gemcita-
bine treatment for ductal adenocarcinoma of the pancreas580
has shown good efficacy. However, the clinical effect was
minimal in patients with advanced solid tumors,576,581 including
metastatic pancreatic cancer.582
In addition, RO4929097, another selective GSI, showed good
anti-tumor activity in preclinical and early trials,583,584 but was not
good for metastatic colorectal cancer,585 metastatic pancreatic

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adenocarcinoma,586 or recurrent platinum-resistant ovarian can-
cer.587 Combinations of RO4929097 with gemcitabine,588 temsir-
olimus,587 cediranib,589 or capecitabine590 in advanced solid
tumors, as well as with bevacizumab in recurrent high-grade
glioma, are well tolerated and have modest clinical benefits.
However, NCT01154452, the combination of RO4929097 with
vismodegib and vismodegib alone for patients with advanced
osteosarcoma, showed no significant difference in a phase Ib trial.
The third oral GSI, PF-03084014, had good efficacy in desmoid
tumors either in phase I or subsequent phase II studies.591
Preliminary evidence of its clinical efficacy was demonstrated in
patients with solid tumors,592 as well as in patients with recurrent
acute T cell lymphoblastic leukemia.593 Other selective GSIs, such
as BMS-906024 (NCT01292655), BMS-986115 (NCT01986218), CB-
103 (NCT03422679), LY3039478 (NCT02836600), and LY900009
(NCT01158404), have also entered the clinical trial stage, and the
results still need to be verified.
DLL4 plays a vital role in regulating tumor angiogenesis.594
Therefore, targeting DLL4 is another strategy to block Notch
signaling, and this is being tested in the clinic. Demcizumab (OMP-
21M18), a humanized IgG2 mAb that targets DLL4 and blocks its
interactions with Notch receptors, was tested in a phase I dose
escalation study with 55 patients with previously treated solid
tumors.595 The results have shown that demcizumab had good
efficacy against solid tumors, but was not good for metastatic
pancreatic cancer treatment when combined with gemcitabine
and Abraxane (NCT02289898). NCT02259582, a combination of
demcizumab with carboplatin and pemetrexed to treat lung
cancers (DENALI study), is ongoing in another phase II study.595
Enoticumab, another fully human IgG1 antibody against DLL4, has
promising activity in phase I clinical trials for advanced solid
malignancies.
Activation of Hh signaling has been implicated in a variety of
cancers.596–598 Activation of Hh signaling in CSCs contributes to
CSC stemness, chemoresistance, and metastatic dissemination.
The Hh signaling pathway mainly regulates target gene expression
via smoothened (SMO)-mediated nuclear transfer of transcription
factors. Three oral SMO antagonists, vismodegib (GDC-0449),
sonidegib (LDE225), and glasdegib (PF-04449913), have been
approved by the Food and Drug Administration (FDA) and show
significant activity in locally advanced and metastatic basal cell
carcinoma, as well as in AML.599–601 Vismodegib was the first
proposed Hh pathway inhibitor in cancer research602 and is
approved by the FDA603 for local or advanced metastatic basal cell
carcinoma treatment.599 Subsequently, phase I and phase II trials
targeting recurrent medulloblastoma have shown that the
progression-free survival (PFS) of Shh-mb patients treated with
vismodegib is longer and more effective than that of non-Shh-mb
patients. Vismodegib even has better activity in patients with
recurrent Shh-mb but not in patients with recurrent non-Shh-
mb.604,605 Vismodegib has also been tested in metastatic color-
ectal cancer,606 pancreatic cancer,607 chondrosarcoma,608
relapsed/refractory NHL, CLL,609 and ovarian cancer.610 Disap-
pointingly, these treatments with vismodegib have not resulted in
better survival.
Sonidegib was the second SMO antagonist approved for the
treatment of locally advanced basal cell carcinoma that recurred
after surgery or radiotherapy and is not suitable for surgery or
radiation therapy.611 In addition, the results of a multicenter,
randomized, double-blind phase II trial have shown that 200 mg
sonidegib for patients with advanced basal cell carcinoma is the
most clinically appropriate dose.600
In a phase I study of a 3 + 3 dose escalation to treat small-cell
lung cancer patients, sonidegib combined with cisplatin and
etoposide sustained PFS in patients with Sox2 amplification.224
These combinations in a phase II trial for patients with recurrent
medulloblastoma resulted in a complete or partial response in
50% of patients612 and have been used for other cancer
Signal Transduction and Targeted Therapy (2020)5:8
Targeting cancer stem cell pathways for cancer therapy
Yang et al.
17
treatments in phase I/II clinical trials, such as NCT02111187 for
prostate cancer, NCT02027376 for breast cancer, and
NCT02195973 for recurrent ovarian cancer.
Glasdegib was the first Hh pathway inhibitor approved for the
treatment of AML in patients older than 75 years or those unable
to use intensive induction chemotherapy601 and showed good
safety and tolerability in a phase I trial for patients with partial
hematologic malignancies in Japan.613 In a phase II trial, glasdegib
combined with cytarabine/daunorubicin had a significant efficacy
in patients with AML, chronic myeloid leukemia (CML) or high-risk
myelodysplastic syndromes.614 Glasdegib combined with low-
dose cytarabine (LDAC) is a potential option for AML patients who
are not suitable for intensive chemotherapy.615 Other selective
SMO inhibitors, including taladegib (LY2940680) and saridegib
(IPI-926), have also entered clinical trials for other cancers. As
single-target agents, these SMO inhibitors have drug resistance
problems. To reduce this problem, some novel inhibitors of
terminal components of Hh signaling pathway are being
developed, such as arsenic trioxide (ATO)616 and GANT-61.617
The Wnt signaling pathway is associated with tumor develop-
ment in breast cancer,618 ovarian cancer,619 esophageal squamous
cell cancer,620 colon cancer,621 prostate cancer,622 and lung
cancer.623 Until now, several drugs aimed at the Wnt signaling
pathway have been in clinical trials, while the majority of Wnt
inhibitors are in preclinical testing. Clinical data from initial trials
have shown that ipafricept (OMP-54f28/FZD8-Fc) is a first-in-class
recombinant fusion protein that antagonizes Wnt signaling.624
However, its role in patients with desmoid cancers and germ cell
cancers is negligible.625 NCT02050178, ipafricept combined with
ab-paclitaxel and gemcitabine in patients with untreated stage IV
pancreatic cancer, NCT02092363, ipafricept combined with
paclitaxel and carboplatin in patients with recurrent platinum-
sensitive ovarian cancer, and NCT02069145, ipafricept combined
with sorafenib in patients with HCC, are currently being
investigated. PRI-724, a β-catenin inhibitor, inhibits the interaction
between β-catenin and its transcriptional coactivators. Safety and
efficacy testing of PRI-724 for patients with advanced myeloid
malignancies (NCT01606579) and advanced or metastatic pan-
creatic cancer (NCT01764477) have been completed in phase I
studies. CWP232291, another inhibitor of β-catenin activity, has
also been shown to be effective for AML (NCT03055286) in a
phase I clinical study and for recurrent or refractory myeloma
(NCT02426723) in a phase I/II clinical study.626 Other Wnt signaling
inhibitors have also been under clinical trial, including LGK974
(NCT02278133), ETC-159 (NCT02521844), and OMP-18R5
(NCT01973309, NCT01957007, and NCT02005315).
In addition, the mitochondrial glycolysis pathway also plays a
key role in regulating the proliferation and apoptosis of CSCs.
Venetoclax, a BCL-2 inhibitor, was initially approved by the FDA
recently and shows good tolerance and activity for AML patients
with adverse reactions.627 Two arachidonate 5-lipoxygenase
inhibitors, VIA-2291 and GSK2190915, might be potent agents
for targeting LSCs in CML,628 as shown in Table 3.
Other abnormal signaling pathways have also been found
in CSCs, such as TGF-β, JAK-STAT, PI3K, and NF-κB. These
signaling pathways are not independent of each other but
rather form a complex signaling network. Agents targeting CSC-
associated signaling pathways in ongoing clinical trials are
listed in Table 3.
Targeting the CSC microenvironment
The CSC microenvironment contributes to the self-renewal and
differentiation of CSCs and regulates CSC fate by providing cues in
the form of secreted factors and cell contact. CXCR4 has been
found in most cancers, especially in CSCs. The most well-
characterized drug-targeting CXCR4 is plerixafor (AMD3100), and
this drug is an effective hematopoietic stem cell mobilizer for
patients with multiple myeloma and NHL.629 AMD3100 treatment
 18
Table 3. Agents targeting CSC-associated signaling pathways and microenvironment in ongoing clinical trials

Drug name Target Condition Phase Sample size NCT number Current status

Hedgehog inhibitors
Vismodegib (GDC-0449) Smoothened Recurrent or refractory medulloblastoma II 31 NCT00939484 Completed, has results
Basal cell carcinoma 28 NCT01700049 Completed, has results
Sarcoma 78 NCT01700049 Completed, has results
Recurrent small-cell lung carcinoma 168 NCT01700049 Completed, has results
Metastatic pancreatic cancer 98 NCT01088815 Completed, has results
Ovarian cancer 104 NCT00739661 Completed, has results
Metastatic colorectal cancer 199 NCT00636610 Completed, has results
Sonidegib (LDE225) Basal cell carcinoma II 10 NCT01350115 Completed, has results
Relapsed medulloblastoma 20 NCT01708174 Completed, has results
Acute myeloid leukemia 70 NCT01826214 Completed, has results
Pancreatic adenocarcinoma 20 NCT01431794 Completed, has results
Advanced or metastatic hepatocellular carcinoma 9 NCT02151864 Completed
Recurrent plasma cell myeloma 28 NCT02086552 Active, not recruiting,
has results
Advanced pancreatic cancer 39 NCT01485744 Active, not recruiting
Advanced breast cancer I 12 NCT02027376 Completed, has results
Glasdegib Acute myeloid leukemia II 255 NCT01546038 Completed, has results
BMS-833923 (XL139) Solid tumors II 12 NCT01413906 Completed
Small-cell lung carcinoma 5 NCT00927875 Completed
Yang et al.
Targeting cancer stem cell pathways for cancer therapy

Metastatic gastric, gastroesophageal, esophageal 39 NCT00909402 Completed

adenocarcinomas
Advanced or metastatic basal cell carcinoma 53 NCT00670189 Completed
Leukemia 70 NCT01357655 Terminated, has results
Taladegib (LY2940680) Localized esophageal or gastroesophageal II 9 NCT02530437 Active, not recruiting
junction cancer
Small-cell lung carcinoma 26 NCT01722292 Terminated, has results
LEQ-506 Solid tumors I 57 NCT01106508 Completed
G-024856 BCC I
Patidegib (IPI-926) Basal cell carcinomas II 36 NCT02828111 Completed, has results
Metastatic or locally advanced chondrosarcoma 105 NCT01310816 Completed
Metastatic pancreatic cancer 122 NCT01130142 Completed
Recurrent head and neck cancer I 9 NCT01255800 Completed
Notch inhibitors
MK-0752 γ-Secretase Advanced breast cancer I 103 NCT00106145 Completed
Pancreatic cancer I 44 NCT01098344 Completed
Metastatic breast cancer I/II 30 NCT00645333 Completed, has results
RO4929097 Recurrent melanoma II 14 NCT01196416 Completed, has results
Advanced or metastatic sarcoma 78 NCT01154452 Completed, has results
Recurrent renal cell carcinoma 12 NCT01141569 Completed, has results

Signal Transduction and Targeted Therapy (2020)5:8

Advanced solid tumors 20 NCT01131234 Completed
Recurrent and/or metastatic epithelial ovarian cancer, 45 NCT01175343 Completed, has results
fallopian tube cancer, or primary peritoneal cancer
Metastatic pancreas cancer 18 NCT01232829 Completed, has results
Recurrent colon cancer 37 NCT01116687 Completed, has results
 Table 3 continued
Drug name Target Condition Phase Sample size NCT number Current status

Recurrent or refractory non-small-cell lung cancer 7 NCT01070927 Completed

Nirogacestat (PF-03084014) Metastatic cancer pancreas II 3 NCT02109445 Terminated, has results
Fibromatosis II 17 NCT01981551 Active, not recruiting
Triple-negative breast neoplasms II 19 NCT02299635 Terminated, has results
LY900009 Advanced cancer I 35 NCT01158404 Completed, has results
Crenigacestat (LY3039478) Pan-Notch Advanced solid tumor I 12 NCT02836600 Active, not recruiting
T cell acute lymphoblastic leukemia, T cell I/II 36 NCT02518113 Completed, has results
lymphoblastic lymphoma
AL101 Adenoid cystic carcinoma II 36 NCT03691207 Recruiting
CB-103 Advanced or metastatic solid tumors and I/II 165 NCT03422679 Recruiting
hematological malignancies
BMS-906024 Advanced or metastatic solid tumors I 94 NCT01292655 Completed
Lymphoblastic leukemia, acute T cell I 31 NCT01363817 Completed
Demcizumab (OMP-21M18) DLL4 Pancreatic cancer II 207 NCT02289898 Completed, has results

Signal Transduction and Targeted Therapy (2020)5:8

Non-squamous, non-small-cell neoplasm of lung II 82 NCT02259582 Completed, has results
Brontictuzumab (OMP-52M51) Adenoid cystic carcinoma Not applicable 1 NCT02662608 Completed, has results
Enoticumab (MEDI528) Advanced solid malignancies I 83 NCT00871559 Completed
MEDI0639 Solid tumors I 58 NCT01577745 Completed, has results
Wnt inhibitors
Ipafricept (OMP-54F28) Wnt receptor Solid tumors I 26 NCT01608867 Completed
Pancreatic cancer I 26 NCT02050178 Completed
Ovarian cancer I 37 NCT02092363 Completed
Hepatocellular cancer I 10 NCT02069145 Completed
Vantictumab (OMP-18R5) Metastatic breast cancer I 37 NCT01973309 Completed
Solid tumors I 35 NCT01345201 Completed
Pancreatic cancer I 30 NCT02005315 Completed
Yang et al.

PRI-724 β-Catenin/CBP Colorectal adenocarcinoma II 0 NCT02413853 Withdrawn

Acute myeloid leukemia 49 NCT01606579 Completed
Solid tumors 23 NCT01302405 Terminated
Advanced pancreatic cancer 20 NCT01764477 Completed
CWP232291 Acute myeloid leukemia I 69 NCT01398462 Completed
Multiple myeloma I 25 NCT02426723 Completed
LGK974 Porcupine Metastatic colorectal cancer I 20 NCT02278133 Completed
Pancreatic cancer I 170 NCT01351103 Recruiting
ETC-1922159 Solid tumors I 65 NCT02521844 Active, not recruiting
Other signaling pathways inhibitors
Galunisertib (LY2157299) TGF-β Prostate cancer II 60 NCT02452008 Recruiting
LY3200882 Colorectal cancer II 31 NCT04031872 Not yet recruiting
AVID200 Malignant solid tumor I 36 NCT03834662 Recruiting
Trabedersen (AP 12009) Pancreatic neoplasms II 62 NCT00844064 Completed
Breast cancer 16 NCT01959490 Completed, has results
Targeting cancer stem cell pathways for cancer therapy

Glioblastoma 141 NCT00431561 Completed

Fresolimumab (GC1008) Non-small-cell lung carcinoma II 60 NCT02581787 Recruiting
Metastatic Breast Cancer 23 NCT01401062 Completed, has results
Carcinoma 29 NCT00356460 Completed
Renal cell
Melanoma
19
 Table 3 continued 20

Drug name Target Condition Phase Sample size NCT number Current status

Vactosertib (TEW-7197) Advanced-stage solid tumors I 35 NCT02160106 Completed

NIS793 Breast cancer I 220 NCT02947165 Recruiting
Lung cancer
Hepatocellular cancer
Ruxolitinib JAK Metastatic breast cancer III 29 NCT01594216 Completed
Myeloproliferative neoplasms 309 NCT00952289 Completed, has results
AZD4205 Advanced non-small-cell lung cancer II 120 NCT03450330 Recruiting
SAR302503 Hematopoietic neoplasm II 97 NCT01523171 Completed
SB1518 JAK/FLT3 Acute myelogenous leukemia II 76 NCT00719836 Completed
PI3K inhibitors
Alpelisib PI3K Advanced breast cancer II 90 NCT03386162 Recruiting
Buparlisib (BKM120) Triple-negative metastatic breast cancer II 50 NCT01629615 Completed
BYL719 Advanced or metastatic gastric cancer I 18 NCT01613950 Completed
SF1126 Advanced or metastatic solid tumors I 44 NCT00907205 Completed
SAR245409 PI3K and mTOR Advanced or metastatic solid tumors I 146 NCT01390818 Completed, has results
EGFR inhibitors
Bevacizumab EGFR Breast cancer I 75 NCT01190345 Completed
Matuzumab (EMD 72000) Esophageal cancer II 72 NCT00215644 Completed, has results
Non-small-cell lung carcinoma 150 NCT00111839 Completed, has results
Metabolism inhibitors
Venetoclax (ABT-199) BCL-2 Acute myelogenous leukemia II 32 NCT01994837 Completed, has results
Yang et al.
Targeting cancer stem cell pathways for cancer therapy

Pegzilarginase Recombinant pegylated Small-cell lung cancer II 84 NCT03371979 Active, not recruiting
arginase
131I-TLX-101 LAT1 Glioblastoma multiforme II 44 NCT03849105 Recruiting
Rifampicin FAS Advanced solid tumors I 36 NCT03077607 Completed, has results
TVB-2640 Advanced breast cancer II 80 NCT03179904 Recruiting
IM156 AMPK Advanced solid tumor I 36 NCT03272256 Recruiting
Telaglenastat Glutaminase Solid tumors II 85 NCT03965845 Recruiting
CB-1158 Arginase Advanced solid tumors II 5 NCT03361228 Completed
Niche inhibitors
Plerixafor (Mozobil) CXCR4 Advanced pancreatic, ovarian, and colorectal cancers I 26 NCT02179970 Completed
BL-8040 Metastatic pancreatic adenocarcinoma II 23 NCT02907099 Active, not recruiting
BKT140 Multiple myeloma II 16 NCT01010880 Completed
BMS-936564 Relapsed/refractory multiple myeloma I 46 NCT01359657 Completed
BMS-936564 Acute myelogenous leukemia I 98 NCT01120457 Completed
LY2510924 Solid tumor I 9 NCT02737072 Terminated, has results
MSX-122 Refractory metastatic or locally advanced solid tumors I 27 NCT00591682 Suspended
USL311 Advanced solid tumors and relapsed/recurrent II 120 NCT02765165 Recruiting
Glioblastoma multiforme
AMD3100 Acute myeloid leukemia II 52 NCT00512252 Completed, has results
Reparixin CXCR1/2 Breast cancer II 20 NCT01861054 Terminated

Signal Transduction and Targeted Therapy (2020)5:8

Defactinib (VS-6063) FAK Non-small-cell lung cancer II 55 NCT01951690 Completed

for relapsed or refractory AML resulted in 46% of patients with
complete remission with or without white count recovery in a
phase I/II study.630 In addition, plerixafor with high-dose
cytarabine and etoposide treatment for children with relapsed
or refractory acute leukemia or myelodysplasia syndrome resulted
in two patients with complete remission and one patient with
incomplete hematologic recovery out of 18 patients in a phase I
study.631 LY2510924, a small cyclic peptide, is a potent and
selective antagonist of CXCR4 and is well tolerated with no serious
adverse events in a phase I trial.632 However, the combination of
LY2510924 with sunitinib for patients with metastatic renal cell
carcinoma has no better effect than sunitinib alone in a phase II
trial.633 The combination of LY2510924 with carboplatin/etoposide
for patients with extensive small-cell lung cancer also had no
significant effect compared with that of carboplatin/etoposide
alone in a phase II study.634 The combination of LY2510924 with
other drugs for gliomas (NCT03746080, NCT01977677, and
NCT01288573) and multiple myeloma (NCT00103662, NCT0122
0375, and NCT00903968) is also under clinical trial.
The microenvironment plays an important role in CSC growth,
and it is also a promising target for treatment. Agents targeting
the microenvironment in ongoing clinical trials are listed in
Table 3.
CSC-directed immunotherapy
In the early twentieth century, Paul Ehrlich first proposed the idea
that an intact immune system suppresses tumor development
(advancing cancer therapy with present and Emerging Immuno-
Oncology Approaches). Based on the understanding of cellular
immune regulation, new methods for cancer treatment have
emerged. In addition to the antibodies against the CSC molecules
mentioned above, some novel anti-CSC immunotherapeutic
approaches, such as immunologic checkpoint blocking or CAR-T
cell therapies, have been developed. Some drugs that target the
immune checkpoint receptors CTLA-4,635 PD-1 (nivolumab,636
pembrolizumab,637 and cemiplimab,638) and PD-L1 (avelumab,639
durvalumab,640 and atezolizumab641) have also been in clinical
trials. I ipilimumab, a CTLA-4 antibody, is approved by the FDA,
and initial clinical results showed good effectiveness in patients
with metastatic melanoma.642 For CAR-T cell therapy, as shown in
Table 4, CD19, CD20, CD22, CD123, EpCAM, and ALDH have been
Targeting cancer stem cell pathways for cancer therapy
Yang et al.
21
used for CSC-directed immunotherapy, and most of them are
recruited.
CONCLUSIONS AND PERSPECTIVES
We can conclude that CSCs are a small population of cancer cells
that have self-renewal capacity and differentiation potential,
thereby conferring tumor relapse, metastasis,643 heterogeneity,644
multidrug resistance,645,646 and radiation resistance.647 Several
pluripotent transcription factors, including Oct4, Sox2, Nanog,
KLF4, and MYC and some intracellular signaling pathways,
including Wnt, NF-κB, Notch, Hh, JAK-STAT, PI3K/AKT/mTOR,
TGF/Smad, and PPAR, as well as extracellular factors, including
vascular niches, hypoxia, TAM, CAF, cancer-associated MSCs, the
ECM, and exosomes, are essential regulators of CSCs. Drugs,
vaccines, antibodies, and CAR-T cells targeting these pathways
have also been developed to target CSCs.648 Importantly, many
clinical trials on CSCs have also been performed and show a
promising future for cancer therapy.
However, there are also multiple hurdles that need to be solved
to effectively eliminate CSCs. First, the characteristics of many
CSCs in specific types of tumors are not well identified.649 Second,
since most studies on CSCs are performed in immune-deficient
mice in the absence of an adaptive immune system, these models
do not recapitulate the biological complexity of tumors in the
clinic.650 Third, CSCs exist in a specific niche that sustains their
survival. However, isolated CSCs are used in most current studies
that lacks a microenvironment.651 Fourth, the environmental
factors in CSC niches are not well understood, and the relationship
between TAMs/CAFs and CSCs has not been well studied.645 Fifth,
since CSCs also share some signaling pathways with normal stem
cells, not all the regulatory factors that contribute to CSCs are
appropriate for use as therapeutic targets in cancer treatment.
Sixth, whether CSCs should be activated or arrested is an open
question in cancer therapy.652 Seventh, novel signaling and more
regulatory levels, such as RNA editing,653 epigenetics,654 and
cellular metabolism,655 should be considered in cancer therapy
because they also contribute to the stemness of CSCs. Eighth,
some inhibitors that target CSC signaling are not very specific, and
so new inhibitors need to be designed.656 Ninth, natural products
that target CSCs should also be studied in the future.657 Finally,

Table 4. CSC-directed immunotherapy in ongoing clinical trials

Trial description Condition Sample size Phase NCT Number Current status

CD19 CAR-T B cell leukemia and lymphoma II 80 NCT03398967 Recruiting

CD123 CAR-T CD122+ myeloid malignancies II 45 NCT02937103 Recruiting
CD22 CAR-T Recurrent or refractory B cell malignancy I/II 45 NCT02794961 Unknown
CD22 CAR-T B-ALL I 15 NCT02650414 Recruiting
CD33 CAR-T Myeloid malignancies I/II 45 NCT02958397 Recruiting
CD33 CAR-T CD32+ acute myeloid leukemia I 11 NCT03126864 Active, not recruiting
CD38 CAR-T B-ALL II 80 NCT03754764 Recruiting
CD138 CAR-T Multiple myeloma II 10 NCT03196414 Recruiting
MUC1 CAR-T/PD-1 KO Advanced esophageal cancer I/II 20 NCT03706326 Recruiting
EGFR IL-12 CAR-T Metastatic colorectal cancer I 20 NCT03542799 Not yet recruiting
MESO CAR-T Refractory–relapsed ovarian cancer I/II 20 NCT03916679 Recruiting
MESO-19 CAR-T Metastatic pancreatic cancer I 4 NCT02465983 Completed
LeY CAR-T Myeloid malignancies I/II 445 NCT02958384 Recruiting
MOv19-BBz CAR -T Recurrent high-grade serous ovarian cancer I 18 NCT03585764 Recruiting
LeY CAR-T Advanced cancer I 30 NCT03851146 Recruiting
EpCAM CAR-T Recurrent breast cancer I 30 NCT02915445 Recruiting
BCMA CAR-T Multiple myeloma II 80 NCT03767751 Recruiting

Signal Transduction and Targeted Therapy (2020)5:8

 Targeting cancer stem cell pathways for cancer therapy
Yang et al.
22
novel ways of targeting the microenvironment of CSCs are also
promising and need to be explored.
ACKNOWLEDGEMENTS
This work was supported by the National Key Research and Development Program of
China (nos. 2016YFC1302204 and 2017YFC1308600), the National Science Founda-
tion of China (nos. 81672502, 81872071, and 81902664) and the Natural Science
Foundation of Chongqing (no. cstc2019jcyj-zdxmX0033).
ADDITIONAL INFORMATION
Competing interests: The authors declare no competing interests.
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